1. Structure of proteins, methods of its determination

STRUCTURE OF PROTEINS, METHODS OF ITS DETERMINATION. PHYSICAL-CHEMICAL PROPERTIES OF SIMPLE AND COMPLEX PROTEINS, PRECIPITATION REACTIONS. STUDING OF THE STRUCTURE AND PHYSICAL-CHEMICAL PROPERTIES OF ENZYMES. MECHANISM OF ENZYME ACTION. VITAMINS AS NUTRITION COMPONENTS: EXOGENIC AND ENDOGENIC HYPOVITAMINOSIS. WATER AND FAT SOLUBLE VITAMINS.

 

Biochemistry is the study of the chemical processes and transformations in living organisms. It deals with the structure and function of cellular components, such as proteins, carbohydrates, lipids, nucleic acids, and other biomolecules. Chemical biology aims to answer many questions arising from biochemistry by using tools developed within synthetic chemistry.

Although there are a vast number of different biomolecules, many are complex and large molecules (called polymers) that are composed of similar repeating subunits (called monomers). Each class of polymeric biomolecule has a different set of subunit types. For example, a protein is a polymer made up of many amino acids. Biochemistry studies the chemical properties of important biological molecules, like proteins, in particular the chemistry of enzyme-catalyzed reactions (enzymes are a type of protein).

The biochemistry of cell metabolism and the endocrine system has been extensively described. Other areas of biochemistry include the genetic code (DNA, RNA), protein synthesis, cell membrane transport, and signal transduction.

This article only discusses terrestrial biochemistry (carbon- and water-based), as all the life forms we know are on Earth. Since life forms alive today are hypothesized by most to have descended from the same common ancestor, they would naturally have similar biochemistries, even for matters that seem to be essentially arbitrary, such as handedness of various biomolecules. It is unknown whether alternative biochemistries are possible or practical.

History of biochemistry

In the 19th century, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur came to the conclusion that this fermentation was catalyzed by a vital force contained within the yeast cells called "ferments", which were thought to function only within living organisms. He wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells."

In 1878 German physiologist Wilhelm Kühne (18371900) coined the term enzyme, which comes from Greek ενζυμον "in leaven", to describe this process. The word enzyme was used later to refer to nonliving substances such as pepsin, and the word ferment used to refer to chemical activity produced by living organisms.

In 1897 Eduard Buchner began to study the ability of yeast extracts to ferment sugar despite the absence of living yeast cells. In a series of experiments at the University of Berlin, he found that the sugar was fermented even when there were no living yeast cells in the mixture. He named the enzyme that brought about the fermentation of sucrose "zymase". In 1907 he received the Nobel Prize in Chemistry "for his biochemical research and his discovery of cell-free fermentation". Following Buchner's example; enzymes are usually named according to the reaction they carry out. Typically the suffix -ase is added to the name of the substrate (e.g., lactase is the enzyme that cleaves lactose) or the type of reaction (e.g., DNA polymerase forms DNA polymers).

Having shown that enzymes could function outside a living cell, the next step was to determine their biochemical nature. Many early workers noted that enzymatic activity was associated with proteins, but several scientists (such as Nobel laureate Richard Willstätter) argued that proteins were merely carriers for the true enzymes and that proteins per se were incapable of catalysis. However, in 1926, James B. Sumner showed that the enzyme urease was a pure protein and crystallized it; Sumner did likewise for the enzyme catalase in 1937. The conclusion that pure proteins can be enzymes was definitively proved by Northrop and Stanley, who worked on the digestive enzymes pepsin (1930), trypsin and chymotrypsin. These three scientists were awarded the 1946 Nobel Prize in Chemistry.

This discovery that enzymes could be crystallized eventually allowed their structures to be solved by x-ray crystallography. This was first done for lysozyme, an enzyme found in tears, saliva and egg whites that digests the coating of some bacteria; the structure was solved by a group led by David Chilton Phillips and published in 1965. This high-resolution structure of lysozyme marked the beginning of the field of structural biology and the effort to understand how enzymes work at an atomic level of detail.

Originally, it was generally believed that life was not subject to the laws of science the way non-life was. It was thought that only living beings could produce the molecules of life (from other, previously existing biomolecules). Then, in 1828, Friedrich Wöhler published a paper about the synthesis of urea, proving that organic compounds can be created artificially. The dawn of biochemistry may have been the discovery of the first enzyme, diastase (today called amylase), in 1833 by Anselme Payen. Eduard Buchner contributed the first demonstration of a complex biochemical process outside of a cell in 1896: alcoholic fermentation in cell extracts of yeast. Although the term biochemistry seems to have been first used in 1882, it is generally accepted that the formal coinage of biochemistry occurred in 1903 by Carl Neuber, a German chemist. Since then, biochemistry has advanced, especially since the mid-20th century, with the development of new techniques such as chromatography, X-ray diffraction, NMR spectroscopy, radioisotopic labelling, electron microscopy and molecular dynamics simulations. These techniques allowed for the discovery and detailed analysis of many molecules and metabolic pathways of the cell, such as glycolysis and the Krebs cycle (citric acid cycle).

Today, the findings of biochemistry are used in many areas, from genetics to molecular biology and from agriculture to medicine.

Characteristics of Enzymes

Enzymes are proteins that catalyze (i.e. accelerate) chemical reactions. In these reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products. Almost all processes in a biological cell need enzymes in order to occur at significant rates. Since enzymes are extremely selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.

Like all catalysts, enzymes work by lowering the activation energy (ΔG) for a reaction, thus dramatically accelerating the rate of the reaction. Most enzyme reaction rates are millions of times faster than those of comparable uncatalyzed reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other catalysts by being much more specific. Enzymes are known to catalyze about 4,000 biochemical reactions. Not all biochemical catalysts are proteins, since some RNA molecules called ribozymes also catalyze reactions.

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Enzymes are protein molecules that are tailored to recognize and bind specific reactants and speed their conversion into products. These proteins are responsible for increasing the rates of all of the many thousand of reaction taking place inside cells.

All enzymatic proteins have several characteristics in common table 1.

Table 1: Characteristics of enzymes proteins

1.

Enzymes combine briefly with reactants during an enzyme-catalyzed reaction.

2.

Enzymes are released unchanged after catalyzing the conversion of reactants to Product

3.

Enzymes are specific in their activity; each enzyme catalyzes the reaction of a single type of molecules or a group of closely related molecules.

4.

Enzymes are saturated by high substrate concentrations.

5.

Many enzymes contain nonproteins groups called cofactors, which contribute to their activity. Inorganic cofactors are all metallic ions. Organic cofactors, called coenzymes, are complex groups derived from vitamins.

6.

Many enzymes are pH and temperature sensitive

The rate of combination and release, known as the turnover number, lies near 1000 per second for most enzymes. Some enzymes have turnover numbers as small as 100 per second or as large as 10 million per second. As a result of enzyme turnover, a relatively small number of enzyme molecules can catalyze a large number of reactant molecules.

The part of an enzyme that combines with substrate molecule is the active site. In most enzymes the active site is located in a cavity or pocket on the enzyme surface, frequently within a cleft marking the boundary between two or more major domains. Within the cleft or pocket, amino acid side groups are situated to fit and bind parts of substrate molecules that are critical to the reaction catalyzed by the enzyme. The active site also separates substrate molecules from the surrounding solutions and place them in environments with unique characteristics, including partial or complete exclusion of water.

How Enzymes Lower the Energy of Activation

The mechanisms by which enzymes lower the energy of activation are still not totally understood. However, the mechanisms are believed to be directly or indirectly related to achievement of what is known as the transition state for a reaction. During any chemical interaction the reactants briefly enter a state in which old chemical bonds are incompletely broken and new ones are incompletely formed. In this transition state electron orbital assume intermediate positions between their locations in the reactants and their positions in the products. The transition state is highly unstable and can easily move in either direction with little change in energy - forward toward products ore back ward toward reactants. In effect, achievement of the transition state places a reacting system in a poised and precariously balanced position at the top of the activation energy barrier.

For example, in the transfer of a phosphate group from one molecule to another, a transition state is set up in which both molecules (shown as X and Y in Figure 2) link to the phosphate group a fraction of a second via transitory bonds (dotted lines). This unstable state can change readily in the direction of either products or unchanged reactants.

Enzymes as Biological Catalysts

 

In cells and organisms most reactions are catalyzed by enzymes, which are regenerated during the course of a reaction. These biological catalysts are physiologically important because they speed up the rates of reactions that would otherwise be too slow to support life. Enzymes increase reaction rates--- sometimes by as much as one millionfold, but more typically by about one thousand fold. Catalysts speed up the forward and reverse reactions proportionately so that, although the magnitude of the rate constants of the forward and reverse reactions is are increased, the ratio of the rate constants remains the same in the presence or absence of enzyme. Since the equilibrium constant is equal to a ratio of rate constants, it is apparent that enzymes and other catalysts have no effect on the equilibrium constant of the reactions they catalyze.

 

Enzymes increase reaction rates by decreasing the amount of energy required to form a complex of reactants that is competent to produce reaction products. This complex is known as the activated state or transition state complex for the reaction. Enzymes and other catalysts accelerate reactions by lowering the energy of the transition state. The free energy required to form an activated complex is much lower in the catalyzed reaction. The amount of energy required to achieve the transition state is lowered; consequently, at any instant a greater proportion of the molecules in the population can achieve the transition state. The result is that the reaction rate is increased.

A number of mechanisms operate to contribute to formation of the transition state. One is bringing reacting molecules into close proximity. Many reactions involve combination or interaction of two or more reactant molecules. For the reaction to take place, the substrate molecule must collide. The required collisions may be rate among reactant molecules suspended in free solution, particularly if the substrates are present in low concentrations. Binding at the active site of an enzyme brings the reactants close together, raising their effective concentration in the active site to many time the concentration in the surrounding solution.

A second contribution mechanism is orienting reactants in positions favoring their interaction. Binding at the active site may bring substrate molecules into an arrangement in which they can collide ate exactly the correct positions and angles required for achievement of the transition state.

The third contributing mechanism is exposing reactant molecules to altered environments that promote their interaction. Enzyme-Substrate Interactions

 

The favored model of enzyme substrate interaction is known as the induced fit model. This model proposes that the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly induce conformational changes in the enzyme that strengthen binding and bring catalytic sites close to substrate bonds to be altered. After binding takes place, one or more mechanisms of catalysis generates transition- state complexes and reaction products. The possible mechanisms of catalysis are four in number:

 

1. Catalysis by Bond Strain: In this form of catalysis, the induced structural rearrangements that take place with the binding of substrate and enzyme ultimately produce strained substrate bonds, which more easily attain the transition state. The new conformation often forces substrate atoms and bulky catalytic groups, such as aspartate and glutamate, into conformations that strain existing substrate bonds.

 

2. Catalysis by Proximity and Orientation: Enzyme-substrate interactions orient reactive groups and bring them into proximity with one another. In addition to inducing strain, groups such as aspartate are frequently chemically reactive as well, and their proximity and orientation toward the substrate thus favors their participation in catalysis.

 

3. Catalysis Involving Proton Donors (Acids) and Acceptors (Bases): Other mechanisms also contribute significantly to the completion of catalytic events initiated by a strain mechanism, for example, the use of glutamate as a general acid catalyst (proton donor).

 

4. Covalent Catalysis: In catalysis that takes place by covalent mechanisms, the substrate is oriented to active sites on the enzymes in such a way that a covalent intermediate forms between the enzyme or coenzyme and the substrate. One of the best-known examples of this mechanism is that involving proteolysis by serine proteases, which include both digestive enzymes (trypsin, chymotrypsin, and elastase) and several enzymes of the blood clotting cascade. These proteases contain an active site serine whose R group hydroxyl forms a covalent bond with a carbonyl carbon of a peptide bond, thereby causing hydrolysis of the peptide bond.

Some reactions, for example, take place more readily in nonpolar environments. Active sites may create such an environment by binding reactants so closely that water molecules are excluded. Another important environmental change is creation of acidic or basic conditions by groups in the active site that release or take up H+ .

Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity; activators are molecules that increase activity. Many drugs and poisons are enzyme inhibitors. Activity is also affected by temperature, pH, and the concentration of substrate. Some enzymes are used commercially, for example, in the synthesis of antibiotics. In addition, some household products use enzymes to speed up biochemical reactions (e.g., enzymes in biological washing powders break down protein or fat stains on clothes; enzymes in meat tenderizers break down proteins, making the meat easier to chew).

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Most enzymes are much larger than the substrates they act on, and only a very small portion of the enzyme (around 34 amino acids) is directly involved in catalysis.

http://www.youtube.com/watch?v=0pJOFze055Y

 

The region that contains these catalytic residues, binds the substrate, and then carries out the reaction is known as the active site. Enzymes can also contain sites that bind cofactors, which are needed for catalysis. Some enzymes also have binding sites for small molecules, which are often direct or indirect products or substrates of the reaction catalyzed.

Structures and mechanisms

Ribbon-diagram showing carbonic anhydrase II. The grey sphere is the zinc cofactor in the active site. Diagram drawn from PDB 1MOO.The activities of enzymes are determined by their three-dimensional structure.

 

This binding can serve to increase or decrease the enzyme's activity, providing a means for feedback regulation. Enzymes are catalysts. Most are proteins. (A few ribonucleoprotein enzymes have been discovered and, for some of these, the catalytic activity is in the RNA part rather than the protein part. Link to discussion of these ribozymes.)

Enzymes bind temporarily to one or more of the reactants of the reaction they catalyze. In doing so, they lower the amount of activation energy needed and thus speed up the reaction.

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/ActivationEnergy.gif

Link to a discussion of free energy (G) and "ΔG".


Examples:

   Catalase. It catalyzes the decomposition of hydrogen peroxide into water and oxygen.

 

 

2H2O2 -> 2H2O + O2

One molecule of catalase can break 40 million molecules of hydrogen peroxide each second.

   Carbonic anhydrase. It is found in red blood cells where it catalyzes the reaction

CO2 + H2O <-> H2CO3

It enables red blood cells to transport carbon dioxide from the tissues to the lungs. One molecule of carbonic anhydrase can process one million molecules of CO2 each second.

   Acetylcholinesterase. It catalyzes the breakdown of the neurotransmitter acetylcholine at several types of synapses as well as at the neuromuscular junction - the specialized synapse that triggers the contraction of skeletal muscle.

One molecule of acetylcholinesterase breaks down 25,000 molecules of acetylcholine each second. This speed makes possible the rapid "resetting" of the synapse for transmission of another nerve impulse.

Like all proteins, enzymes are made as long, linear chains of amino acids that fold to produce a three-dimensional product. Each unique amino acid sequence produces a unique structure, which has unique properties. Individual protein chains may sometimes group together to form a protein complex. Most enzymes can be denaturedthat is, unfolded and inactivatedby heating, which destroys the three-dimensional structure of the protein. Depending on the enzyme, denaturation may be reversible or irreversible.

 

Specificity

Enzymes are usually very specific as to which reactions they catalyze and the substrates that are involved in these reactions. Complementary shape, charge and hydrophilic/hydrophobic characteristics of enzymes and substrates are responsible for this specificity. Enzymes can also show impressive levels of stereospecificity, regioselectivity and chemoselectivity.

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Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome. These enzymes have "proof-reading" mechanisms. Here, an enzyme such as DNA polymerase catalyses a reaction in a first step and then checks that the product is correct in a second step. This two-step process results in average error rates of less than 1 error in 100 million reactions in high-fidelity mammalian polymerases. Similar proofreading mechanisms are also found in RNA polymerase, aminoacyl tRNA synthetases and ribosomes.

Some enzymes that produce secondary metabolites are described as promiscuous, as they can act on a relatively broad range of different substrates. It has been suggested that this broad substrate specificity is important for the evolution of new biosynthetic pathways.

 

"Lock and key" model

Enzymes are very specific, and it was suggested by Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model. However, while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve.

 

 

Induced fit model

Diagrams to show the induced fit hypothesis of enzyme action.In 1958 Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible structures, the active site can be reshaped by interactions with the substrate as the substrate interacts with the enzyme. As a result, the substrate does not simply bind to a rigid active site, the amino acid side chains which make up the active site are molded into the precise positions that enable the enzyme to perform its catalytic function. In some cases, such as glycosidases, the substrate molecule also changes shape slightly as it enters the active site.

 

 

nzymes can act in several ways, all of which lower ΔG:

 

Lowering the activation energy by creating an environment in which the transition state is stabilized (e.g. straining the shape of a substrate - by binding the transition-state conformation of the substrate/product molecules, the enzyme distorts the bound substrate(s) into their transition state form, thereby reducing the amount of energy required to complete the transition).

Providing an alternative pathway (e.g. temporarily reacting with the substrate to form an intermediate ES Complex which would be impossible in the absence of the enzyme).

Reducing the reaction entropy change by bringing substrates together in the correct orientation to react. Considering ΔH alone overlooks this effect.

 

Dynamics and function

Recent investigations have provided new insights into the connection between internal dynamics of enzymes and their mechanism of catalysis. An enzyme's internal dynamics are described as the movement of internal parts (e.g. amino acids, a group of amino acids, a loop region, an alpha helix, neighboring beta-sheets or even entire domain) of these biomolecules, which can occur at various time-scales ranging from femtoseconds to seconds. Networks of protein residues throughout an enzyme's structure can contribute to catalysis through dynamic motions. Protein motions are vital to many enzymes, but whether small and fast vibrations or larger and slower conformational movements are more important depends on the type of reaction involved. These new insights also have implications in understanding allosteric effects, producing designer enzymes and developing new drugs.

 

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Cofactors and coenzymes

Role of Coenzymes

 

The functional role of coenzymes is to act as transporters of chemical groups from one reactant to another. The chemical groups carried can be as simple as the hydride ion (H+ + 2e-) carried by NAD or the mole of hydrogen carried by FAD; or they can be even more complex than the amine (-NH2) carried by pyridoxal phosphate.

Since coenzymes are chemically changed as a consequence of enzyme action, it is often useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different holoenzymes. In all cases, the coenzymes donate the carried chemical grouping to an acceptor molecule and are thus regenerated to their original form. This regeneration of coenzyme and holoenzyme fulfills the definition of an enzyme as a chemical catalyst, since (unlike the usual substrates, which are used up during the course of a reaction) coenzymes are generally regenerated.

 

Enzyme Relative to Substrate Type

Although enzymes are highly specific for the kind of reaction they catalyze, the same is not always true of substrates they attack. For example, while succinic dehydrogenase (SDH) always catalyzes an oxidation-reduction reaction and its substrate is invariably succinic acid, alcohol dehydrogenase (ADH) always catalyzes oxidation-reduction reactions but attacks a number of different alcohols, ranging from methanol to butanol. Generally, enzymes having broad substrate specificity are most active against one particular substrate. In the case of ADH, ethanol is the preferred substrate.

 

Enzymes also are generally specific for a particular steric configuration (optical isomer) of a substrate. Enzymes that attack D sugars will not attack the corresponding L isomer. Enzymes that act on L amino acids will not employ the corresponding D optical isomer as a substrate. The enzymes known as racemases provide a striking exception to these generalities; in fact, the role of racemases is to convert D isomers to L isomers and vice versa. Thus racemases attack both D and L forms of their substrate.

 

As enzymes have a more or less broad range of substrate specificity, it follows that a given substrate may be acted on by a number of different enzymes, each of which uses the same substrate(s) and produces the same product(s). The individual members of a set of enzymes sharing such characteristics are known as isozymes. These are the products of genes that vary only slightly; often, various isozymes of a group are expressed in different tissues of the body. The best studied set of isozymes is the lactate dehydrogenase (LDH) system. LDH is a tetrameric enzyme composed of all possible arrangements of two different protein subunits; the subunits are known as H (for heart) and M (for skeletal muscle). These subunits combine in various combinations leading to 5 distinct isozymes. The all H isozyme is characteristic of that from heart tissue, and the all M isozyme is typically found in skeletal muscle and liver. These isozymes all catalyze the same chemical reaction, but they exhibit differing degrees of efficiency. The detection of specific LDH isozymes in the blood is highly diagnostic of tissue damage such as occurs during cardiac infarct.

Enzyme cofactors

Many enzymes require the presence of an additional, nonprotein, cofactor.

   Some of these are metal ions such as Zn2+ (the cofactor for carbonic anhydrase), Cu2+, Mn2+, K+, and Na+.

   Some cofactors are small organic molecules called coenzymes. The B vitamins

o                          thiamine (B1)

o                          riboflavin (B2) and

o                          nicotinamide

are precursors of coenzymes.

Coenzymes may be covalently bound to the protein part (called the apoenzyme) of enzymes as a prosthetic group. Others bind more loosely and, in fact, may bind only transiently to the enzyme as it performs its catalytic act.

LysozymeTarget

 

 

LysozymeActionLysozyme: a model of enzyme action

A number of lysozymes are found in nature; in human tears and egg white, for examples. The enzyme is antibacterial because it degrades the polysaccharide that is found in the cell walls of many bacteria. It does this by catalyzing the insertion of a water molecule at the position indicated by the red arrow. This hydrolysis breaks the chain at that point.

The bacterial polysaccharide consists of long chains of alternating amino sugars:

   N-acetylglucosamine (NAG)

   N-acetylmuramic acid (NAM)

These hexose units resemble glucose except for the presence of the side chains containing amino groups.

Lysozyme is a globular protein with a deep cleft across part of its surface. Six hexoses of the substrate fit into this cleft.

   With so many oxygen atoms in sugars, as many as 14 hydrogen bonds form between the six amino sugars and certain amino acid R groups such as Arg-114, Asn-37, Asn-44, Trp-62, Trp-63, and Asp-101.

   Some hydrogen bonds also form with the C=O groups of several peptide bonds.

   In addition, hydrophobic interactions may help hold the substrate in position.

X-ray crystallography has shown that as lysozyme and its substrate unite, each is slightly deformed. The fourth hexose in the chain (ring #4) becomes twisted out of its normal position. This imposes a strain on the C-O bond on the ring-4 side of the oxygen bridge between rings 4 and 5. It is just at this point that the polysaccharide is broken. A molecule of water is inserted between these two hexoses, which breaks the chain. Here, then, is a structural view of what it means to lower activation energy. The energy needed to break this covalent bond is lower now that the atoms connected by the bond have been distorted from their normal position.

As for lysozyme itself, binding of the substrate induces a small (~0.75Å) movement of certain amino acid residues so the cleft closes slightly over its substrate. So the "lock" as well as the "key" changes shape as the two are brought together. (This is sometimes called "induced fit".)

The amino acid residues in the vicinity of rings 4 and 5 provide a plausible mechanism for completing the catalytic act. Residue 35, glutamic acid (Glu-35), is about 3Å from the -O- bridge that is to be broken. The free carboxyl group of glutamic acid is a hydrogen ion donor and available to transfer H+ to the oxygen atom. This would break the already-strained bond between the oxygen atom and the carbon atom of ring 4.

Now having lost an electron, the carbon atom acquires a positive charge. Ionized carbon is normally very unstable, but the attraction of the negatively-charged carboxyl ion of Asp-52 could stabilize it long enough for an -OH ion (from a spontaneously dissociated water molecule) to unite with the carbon. Even at pH 7, water spontaneously dissociates to produce H+ and OH- ions. The hydrogen ion (H+) left over can replace that lost by Glu-35.

In either case, the chain is broken, the two fragments separate from the enzyme, and the enzyme is free to attach to a new location on the bacterial cell wall and continue its work of digesting it.

 

Cofactors

Some enzymes do not need any additional components to show full activity. However, others require non-protein molecules to be bound for activity. Cofactors can be either inorganic (e.g., metal ions and iron-sulfur clusters) or organic compounds, (e.g., flavin and heme). Organic cofactors (coenzymes) are usually prosthetic groups, which are tightly bound to the enzymes that they assist. These tightly-bound cofactors are distinguished from other coenzymes, such as NADH, since they are not released from the active site during the reaction.

 

An example of an enzyme that contains a cofactor is carbonic anhydrase, and is shown in the ribbon diagram above with a zinc cofactor bound in its active site. These tightly-bound molecules are usually found in the active site and are involved in catalysis. For example, flavin and heme cofactors are often involved in redox reactions.

 

Enzymes that require a cofactor but do not have one bound are called apoenzymes. An apoenzyme together with its cofactor(s) is called a holoenzyme (i.e., the active form). Most cofactors are not covalently attached to an enzyme, but are very tightly bound. However, organic prosthetic groups can be covalently bound (e.g., thiamine pyrophosphate in the enzyme pyruvate dehydrogenase).

 

Coenzymes

Space-filling model of the coenzyme NADH Coenzymes are small molecules that transport chemical groups from one enzyme to another. Some of these chemicals such as riboflavin, thiamine and folic acid are vitamins, this is when these compounds cannot be made in the body and must be acquired from the diet. The chemical groups carried include the hydride ion (H-) carried by NAD or NADP+, the acetyl group carried by coenzyme A, formyl, methenyl or methyl groups carried by folic acid and the methyl group carried by S-adenosylmethionine.

 

Since coenzymes are chemically changed as a consequence of enzyme action, it is useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different enzymes. For example, about 700 enzymes are known to use the coenzyme NADH.

 

 

Enzymes_pH_temp

Coenzymes are usually regenerated and their concentrations maintained at a steady level inside the cell: for example, NADPH is regenerated through the pentose phosphate pathway and S-adenosylmethionine by methionine adenosyltransferase.

 

Factors Affecting Enzyme Action

The activity of enzymes is strongly affected by changes in pH and temperature. Each enzyme works best at a certain pH (left graph) and temperature (right graph), its activity decreasing at values above and below that point. This is not surprising considering the importance of

   tertiary structure (i.e. shape) in enzyme function and

   noncovalent forces, e.g., ionic interactions and hydrogen bonds, in determining that shape.

Examples:

   the protease pepsin works best as a pH of 1-2 (found in the stomach) while

   the protease trypsin is inactive at such a low pH but very active at a pH of 8 (found in the small intestine as the bicarbonate of the pancreatic fluid neutralizes the arriving stomach contents).

Changes in pH alter the state of ionization of charged amino acids (e.g., Asp, Lys) that may play a crucial role in substrate binding and/or the catalytic action itself. Without the unionized -COOH group of Glu-35 and the ionized -COO- of Asp-52, the catalytic action of lysozyme would cease.

 

MolecularEnergies

Hydrogen bonds are easily disrupted by increasing temperature. This, in turn, may disrupt the shape of the enzyme so that its affinity for its substrate diminishes. The ascending portion of the temperature curve (red arrow in right-hand graph above) reflects the general effect of increasing temperature on the rate of chemical reactions (graph at left). The descending portion of the curve above (blue arrow) reflects the loss of catalytic activity as the enzyme molecules become denatured at high temperatures.

Regulation of Enzyme Activity

Several mechanisms work to make enzyme activity within the cell efficient and well-coordinated.

EnzymePathControl

 

 

Anchoring enzymes in membranes

Many enzymes are inserted into cell membranes, for examples,

   the plasma membrane

   the membranes of mitochondria and chloroplasts

   the endoplasmic reticulum

   the nuclear envelope

These are locked into spatial relationships that enable them to interact efficiently.

Inactive precursors

Enzymes, such as proteases, that can attack the cell itself are inhibited while within the cell that synthesizes them. For example, pepsin is synthesized within the chief cells (in gastric glands) as an inactive precursor, pepsinogen. Only when exposed to the low pH outside the cell is the inhibiting portion of the molecule removed and active pepsin produced.

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Feedback Inhibition

FeedbackInhibition

 

If the product of a series of enzymatic reactions, e.g., an amino acid, begins to accumulate within the cell, it may specifically inhibit the action of the first enzyme involved in its synthesis (red bar). Thus further production of the enzyme is halted.

Precursor Activation

The accumulation of a substance within a cell may specifically activate (blue arrow) an enzyme that sets in motion a sequence of reactions for which that substance is the initial substrate. This reduces the concentration of the initial substrate.

In the case if feedback inhibition and precursor activation, the activity of the enzyme is being regulated by a molecule which is not its substrate. In these cases, the regulator molecule binds to the enzyme at a different site than the one to which the substrate binds. When the regulator binds to its site, it alters the shape of the enzyme so that its activity is changed. This is called an allosteric effect.

   In feedback inhibition, the allosteric effect lowers the affinity of the enzyme for its substrate.

   In precursor activation, the regulator molecule increases the affinity of the enzyme in the series for its substrate.If, for example, ample quantities of an amino acid are already available to the cell from its extracellular fluid, synthesis of the enzymes that would enable the cell to produce that amino acid for itself is shut down.

Conversely, if a new substrate is made available to the cell, it may induce the synthesis of the enzymes needed to cope with it. Yeast cells, for example, do not ordinarily metabolize lactose and no lactase can be detected in them. However, if grown in a medium containing lactose, they soon begin synthesizing lactase - by transcribing and translating the necessary gene(s) - and so can begin to metabolize the sugar.

. coli also has a mechanism which regulates enzyme synthesis by controlling translation of a needed messenger RNA..

Factors Affecting Enzymes


Thermodynamics

Main aticles: Activation energy, Thermodynamic equilibrium, and Chemical equilibrium

Diagram of a catalytic reaction, showing the energy niveau at each stage of the reaction. The substrates usually need a large amount of energy to reach the transition state, which then decays into the end product. The enzyme stabilizes the transition state, reducing the energy needed to form this species and thus reducing the energy required to form products.As all catalysts, enzymes do not alter the position of the chemical equilibrium of the reaction. Usually, in the presence of an enzyme, the reaction runs in the same direction as it would without the enzyme, just more quickly. However, in the absence of the enzyme, other possible uncatalyzed, "spontaneous" reactions might lead to different products, because in those conditions this different product is formed faster.

Furthermore, enzymes can couple two or more reactions, so that a thermodynamically favorable reaction can be used to "drive" a thermodynamically unfavorable one. For example, the hydrolysis of ATP is often used to drive other chemical reactions.

Enzymes catalyze the forward and backward reactions equally. They do not alter the equilibrium itself, but only the speed at which it is reached. For example, carbonic anhydrase catalyzes its reaction in either direction depending on the concentration of its reactants.

(in tissues; high CO2 concentration)

(in lungs; low CO2 concentration)

Nevertheless, if the equilibrium is greatly displaced in one direction, that is, in a very exergonic reaction, the reaction is effectively irreversible. Under these conditions the enzyme will, in fact, only catalyze the reaction in the thermodynamically allowed direction.

Kinetics

 

Mechanism for a single substrate enzyme catalyzed reaction. The enzyme (E) binds a substrate (S) and produces a product (P).Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. The rate data used in kinetic analyses are obtained from enzyme assays. In 1913 Leonor Michaelis and Maud Menten proposed a quantitative theory of enzyme kinetics, which is referred to as Michaelis-Menten kinetics. Their work was further developed by G. E. Briggs and J. B. S. Haldane, who derived kinetic equations that are still widely used today.

 

Michaelis-Menton Kinetics

In typical enzyme-catalyzed reactions, reactant and product concentrations are usually hundreds or thousands of times greater than the enzyme concentration. Consequently, each enzyme molecule catalyzes the conversion to product of many reactant molecules. In biochemical reactions, reactants are commonly known as substrates. The catalytic event that converts substrate to product involves the formation of a transition state, and it occurs most easily at a specific binding site on the enzyme. This site, called the catalytic site of the enzyme, has been evolutionarily structured to provide specific, high-affinity binding of substrate(s) and to provide an environment that favors the catalytic events. The complex that forms, when substrate(s) and enzyme combine, is called the enzyme substrate (ES) complex. Reaction products arise when the ES complex breaks down releasing free enzyme.

 

Between the binding of substrate to enzyme, and the reappearance of free enzyme and product, a series of complex events must take place. At a minimum an ES complex must be formed; this complex must pass to the transition state (ES*); and the transition state complex must advance to an enzyme product complex (EP). The latter is finally competent to dissociate to product and free enzyme. The series of events can be shown thus:

E + S <---> ES <---> ES* <---> EP <---> E + P

The kinetics of simple reactions like that above were first characterized by biochemists Michaelis and Menten. The concepts underlying their analysis of enzyme kinetics continue to provide the cornerstone for understanding metabolism today, and for the development and clinical use of drugs aimed at selectively altering rate constants and interfering with the progress of disease states. The Michaelis-Menten equation is a quantitative description of the relationship among the rate of an enzyme- catalyzed reaction [v1], the concentration of substrate [S] and two constants, Vmax and Km (which are set by the particular equation). The symbols used in the Michaelis-Menton equation refer to the reaction rate [v1], maximum reaction rate (Vmax), substrate concentration [S] and the Michaelis-Menton constant (Km).

 

The Michaelis-Menten equation can be used to demonstrate that at the substrate concentration that produces exactly half of the maximum reaction rate, i.e.,1/2 Vmax, the substrate concentration is numerically equal to Km. This fact provides a simple yet powerful bioanalytical tool that has been used to characterize both normal and altered enzymes, such as those that produce the symptoms of genetic diseases. Rearranging the Michaelis-Menton equation leads to:

From this equation it should be apparent that when the substrate concentration is half that required to support the maximum rate of reaction, the observed rate, v1, will, be equal to Vmax divided by 2; in other words, v1 = [Vmax/2]. At this substrate concentration Vmax/v1 will be exactly equal to 2, with the result that:

[S](1) = Km

The latter is an algebraic statement of the fact that, for enzymes of the Michaelis-Menten type, when the observed reaction rate is half of the maximum possible reaction rate, the substrate concentration is numerically equal to the Michaelis-Menten constant. In this derivation, the units of Km are those used to specify the concentration of S, usually Molarity.

 

The Michaelis-Menten equation has the same form as the equation for a rectangular hyperbola; graphical analysis of reaction rate (v) versus substrate concentration [S] produces a hyperbolic rate plot.

 

Plot of substrate concentration versus reaction velocity

The key features of the plot are marked by points A, B and C. At high substrate concentrations the rate represented by point C the rate of the reaction is almost equal to Vmax, and the difference in rate at nearby concentrations of substrate is almost negligible. If the Michaelis-Menten plot is extrapolated to infinitely high substrate concentrations, the extrapolated rate is equal to Vmax. When the reaction rate becomes independent of substrate concentration, or nearly so, the rate is said to be zero order. (Note that the reaction is zero order only with respect to this substrate. If the reaction has two substrates, it may or may not be zero order with respect to the second substrate). The very small differences in reaction velocity at substrate concentrations around point C (near Vmax) reflect the fact that at these concentrations almost all of the enzyme molecules are bound to substrate and the rate is virtually independent of substrate, hence zero order. At lower substrate concentrations, such as at points A and B, the lower reaction velocities indicate that at any moment only a portion of the enzyme molecules are bound to the substrate. In fact, at the substrate concentration denoted by point B, exactly half the enzyme molecules are in an ES complex at any instant and the rate is exactly one half of Vmax. At substrate concentrations near point A the rate appears to be directly proportional to substrate concentration, and the reaction rate is said to be first order.

 

Inhibition of Enzyme Catalyzed Reactions

 

To avoid dealing with curvilinear plots of enzyme catalyzed reactions, biochemists Lineweaver and Burk introduced an analysis of enzyme kinetics based on the following rearrangement of the Michaelis-Menten equation:

 

[1/v] = [Km (1)/ Vmax[S] + (1)/Vmax]

 

Plots of 1/v versus 1/[S] yield straight lines having a slope of Km/Vmax and an intercept on the ordinate at 1/Vmax.

 

A Lineweaver-Burk Plot

An alternative linear transformation of the Michaelis-Menten equation is the Eadie-Hofstee transformation:

 

v/[S] = -v [1/Km] + [Vmax/Km]

 

and when v/[S] is plotted on the y-axis versus v on the x-axis, the result is a linear plot with a slope of -1/Km and the value Vmax/Km as the intercept on the y-axis and Vmax as the intercept on the x-axis.

Both the Lineweaver-Burk and Eadie-Hofstee transformation of the Michaelis-Menton equation are useful in the analysis of enzyme inhibition. Since most clinical drug therapy is based on inhibiting the activity of enzymes, analysis of enzyme reactions using the tools described above has been fundamental to the modern design of pharmaceuticals. Well- known examples of such therapy include the use of methotrexate in cancer chemotherapy to semi-selectively inhibit DNA synthesis of malignant cells, the use of aspirin to inhibit the synthesis of prostaglandins which are at least partly responsible for the aches and pains of arthritis, and the use of sulfa drugs to inhibit the folic acid synthesis that is essential for the metabolism and growth of disease-causing bacteria. In addition, many poisons, such as cyanide, carbon monoxide and polychlorinated biphenols (PCBs). produce their life- threatening effects by means of enzyme inhibition.

 

The major contribution of Michaelis and Menten was to think of enzyme reactions in two stages. In the first, the substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. This is sometimes called the Michaelis-Menten complex in their honor. The enzyme then catalyzes the chemical step in the reaction and releases the product.

Saturation curve for an enzyme reaction showing the relation between the substrate concentration (S) and rate (v).Enzymes can catalyze up to several million reactions per second. For example, the reaction catalyzed by orotidine 5'-phosphate decarboxylase will consume half of its substrate in 78 million years if no enzyme is present. However, when the decarboxylase is added, the same process takes just 25 milliseconds. Enzyme rates depend on solution conditions and substrate concentration. Conditions that denature the protein abolish enzyme activity, such as high temperatures, extremes of pH or high salt concentrations, while raising substrate concentration tends to increase activity. To find the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of product formation is seen. This is shown in the saturation curve, shown on the right. Saturation happens because, as substrate concentration increases, more and more of the free enzyme is converted into the substrate-bound ES form. At the maximum velocity (Vmax) of the enzyme, all enzyme active sites are saturated with substrate, and the amount of ES complex is the same as the total amount of enzyme.

However, Vmax is only one kinetic constant of enzymes. The amount of substrate needed to achieve a given rate of reaction is also important. This is given by the Michaelis-Menten constant (Km), which is the substrate concentration required for an enzyme to reach one-half its maximum velocity. Each enzyme has a characteristic Km for a given substrate, and this can show how tight the binding of the substrate is to the enzyme. Another useful constant is kcat, which is the number of substrate molecules handled by one active site per second.

The defficiency of an enzyme can be expressed in terms of kcat/Km. This is also called the specificity constant and incorporates the rate constants for all steps in the reaction. Because the specificity constant reflects both affinity and catalytic ability, it is useful for comparing different enzymes against each other, or the same enzyme with different substrates. The theoretical maximum for the specificity constant is called the diffusion limit and is about 108 to 109 (M-1 s-1). At this point every collision of the enzyme with its substrate will result in catalysis, and the rate of product formation is not limited by the reaction rate but by the diffusion rate. Enzymes with this property are called catalytically perfect or kinetically perfect. Example of such enzymes are triose-phosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, ß-lactamase, and superoxide dismutase.

 

Some enzymes operate with kinetics which are faster than diffusion rates, which would seem to be impossible. Several mechanisms have been invoked to explain this phenomenon. Some proteins are believed to accelerate catalysis by drawing their substrate in and pre-orienting them by using dipolar electric fields. Other models invoke a quantum-mechanical tunneling explanation, whereby a proton or an electron can tunnel through activation barriers, although for proton tunneling this model remains somewhat controversial. Quantum tunneling for protons has been observed in tryptamine. This suggests that enzyme catalysis may be more accurately characterized as "through the barrier" rather than the traditional model, which requires substrates to go "over" a lowered energy barrier.

Enzyme inhibitors fall into two broad classes: those causing irreversible inactivation of enzymes and those whose inhibitory effects can be reversed. Inhibitors of the first class usually cause an inactivating, covalent modification of enzyme structure. Cyanide is a classic example of an irreversible enzyme inhibitor: by covalently binding mitochondrial cytochrome oxidase, it inhibits all the reactions associated with electron transport. The kinetic effect of irreversible inhibitors is to decrease the concentration of active enzyme, thus decreasing the maximum possible concentration of ES complex. Since the limiting enzyme reaction rate is often k2[ES], it is clear that under these circumstances the reduction of enzyme concentration will lead to decreased reaction rates. Note that when enzymes in cells are only partially inhibited by irreversible inhibitors, the remaining unmodified enzyme molecules are not distinguishable from those in untreated cells; in particular, they have the same turnover number and the same Km. Turnover number, related to Vmax, is defined as the maximum number of moles of substrate that can be converted to product per mole of catalytic site per second. Irreversible inhibitors are usually considered to be poisons and are generally unsuitable for therapeutic purposes.

 

Reversible inhibitors can be divided into two main categories; with a third category, uncompetitive inhibitors, rarely encountered.

Inhibitor Type

Binding Site on Enzyme

Kinetic effect

Competitive Inhibitor

Specifically at the catalytic site, where it competes with substrate for binding in a dynamic equilibrium- like process. Inhibition is reversible by substrate.

Vmax is unchanged; Km, as defined by [S] required for 1/2 maximal activity, is increased.

Noncompetitive Inhibitor

Binds E or ES complex other than at the catalytic site. Substrate binding unaltered, but ESI complex cannot form products. Inhibition cannot be reversed by substrate.

Km appears unaltered; Vmax is decreased proportionately to inhibitor concentration.

Uncompetitive Inhibitor

Binds only to ES complexes at locations other than the catalytic site. Substrate binding modifies enzyme structure, making inhibitor- binding site available. Inhibition cannot be reversed by substrate.

Apparent Vmax decreased; Km, as defined by [S] required for 1/2 maximal activity, is decreased.

 

Inhibitor Type

Specifically at the catalytic site, where it competes with substrate for binding in a dynamic equilibrium- like process. Inhibition is reversible by substrate.

Vmax is unchanged; Km, as defined by [S] required for 1/2 maximal activity, is increased.

 

http://www.youtube.com/watch?v=0pJOFze055Y

 

Noncompetitive Inhibitor

Binds E or ES complex other than at the catalytic site. Substrate binding unaltered, but ESI complex cannot form products. Inhibition cannot be reversed by substrate.

Km appears unaltered; Vmax is decreased proportionately to inhibitor concentration.

Uncompetitive Inhibitor

Binds only to ES complexes at locations other than the catalytic site. Substrate binding modifies enzyme structure, making inhibitor- binding site available. Inhibition cannot be reversed by substrate.

Apparent Vmax decreased; Km, as defined by [S] required for 1/2 maximal activity, is decreased.

The hallmark of all the reversible inhibitors is that when the inhibitor concentration drops, enzyme activity is regenerated. Usually these inhibitors bind to enzymes by non-covalent forces and the inhibitor maintains a reversible equilibrium with the enzyme. The equilibrium constant for the dissociation of enzyme inhibitor complexes is known as KI:

 

KI = [E][I]/[E--I--complex]

The importance of KI is that in all enzyme reactions where substrate, inhibitor and enzyme interact, the normal Km and or Vmax for substrate enzyme interaction appear to be altered. These changes are a consequence of the influence of KI on the overall rate equation for the reaction. The effects of KI are best observed in Lineweaver-Burk plots.

Probably the best known reversible inhibitors are competitive inhibitors, which always bind at the catalytic or active site of the enzyme. Most drugs that alter enzyme activity are of this type. Competitive inhibitors are especially attractive as clinical modulators of enzyme activity because they offer two routes for the reversal of enzyme inhibition, while other reversible inhibitors offer only one. First, as with all kinds of reversible inhibitors, a decreasing concentration of the inhibitor reverses the equilibrium regenerating active free enzyme. Second, since substrate and competitive inhibitors both bind at the same site they compete with one another for binding .

 

Raising the concentration of substrate (S), while holding the concentration of inhibitor constant, provides the second route for reversal of competitive inhibition. The greater the proportion of substrate, the greater the proportion of enzyme present in competent ES complexes.

As noted earlier, high concentrations of substrate can displace virtually all competitive inhibitor bound to active sites. Thus, it is apparent that Vmax should be unchanged by competitive inhibitors. This characteristic of competitive inhibitors is reflected in the identical vertical-axis intercepts of Lineweaver-Burk plots, with and without inhibitor.

Since attaining Vmax requires appreciably higher substrate concentrations in the presence of competitive inhibitor, Km (the substrate concentration at half maximal velocity) is also higher, as demonstrated by the differing negative intercepts on the horizontal axis in panel B.

 

Analogously, panel C illustrates that noncompetitive inhibitors appear to have no effect on the intercept at the x-axis implying that noncompetitive inhibitors have no effect on the Km of the enzymes they inhibit. Since noncompetitive inhibitors do not interfere in the equilibration of enzyme, substrate and ES complexes, the Km's of Michaelis-Menten type enzymes are not expected to be affected by noncompetitive inhibitors, as demonstrated by x-axis intercepts in panel C. However, because complexes that contain inhibitor (ESI) are incapable of progressing to reaction products, the effect of a noncompetitive inhibitor is to reduce the concentration of ES complexes that can advance to product. Since Vmax = k2[Etotal], and the concentration of competent Etotal is diminished by the amount of ESI formed, noncompetitive inhibitors are expected to decrease Vmax, as illustrated by the y-axis intercepts in panel C.

 

A corresponding analysis of uncompetitive inhibition leads to the expectation that these inhibitors should change the apparent values of Km as well as Vmax. Changing both constants leads to double reciprocal plots, in which intercepts on the x and y axes are proportionately changed; this leads to the production of parallel lines in inhibited and uninhibited reactions.

 

Inhibition

Competitive inhibitors bind reversibly to the enzyme, preventing the binding of substrate. On the other hand, binding of substrate prevents binding of the inhibitor. Substrate and inhibitor compete for the enzyme.Main article: Enzyme inhibitor

Enzyme reaction rates can be decreased by various types of enzyme inhibitors.

 

Reversible inhibitors

In competitive inhibition the inhibitor binds to the substrate binding site (figure right, top, thus preventing substrate from binding (EI complex). Often competitive inhibitors strongly resemble the real substrate of the enzyme. For example, methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase, which catalyzes the reduction of dihydrofolate to tetrahydrofolate. The similarity between the structures of folic acid and this drug are shown in the figure to the right bottom.

 

Non-competitive inhibition

In order to do its work, an enzyme must unite - even if ever so briefly - with at least one of the reactants. In most cases, the forces that hold the enzyme and its substrate are noncovalent, an assortment of:

   hydrogen bonds

   ionic interactions

   and hydrophobic interactions

Link to discussion of the noncovalent forces that hold macromolecules enzyme_substrate

 

together.

Most of these interactions are weak and especially so if the atoms involved are farther than about one angstrom from each other. So successful binding of enzyme and substrate requires that the two molecules be able to approach each other closely over a fairly broad surface. Thus the analogy that a substrate molecule binds its enzyme like a key in a lock.

 

malonic

 

This requirement for complementarity in the configuration of substrate and enzyme explains the remarkable specificity of most enzymes. Generally, a given enzyme is able to catalyze only a single chemical reaction or, at most, a few reactions involving substrates sharing the same general structure.

The necessity for a close, if brief, fit between enzyme and substrate explains the phenomenon of competitive inhibition.

It catalyzes the oxidation (by the removal of two hydrogen atoms) of succinic acid (a). If one adds malonic acid to cells, or to a test tube mixture of succinic acid and the enzyme, the action of the enzyme is strongly inhibited. This is because the structure of malonic acid allows it to bind to the same site on the enzyme (b). But there is no oxidation so no speedy release of products. The inhibition is called competitive because if you increase the ratio of succinic to malonic acid in the mixture, you will gradually restore the rate of catalysis. At a 50:1 ratio, the two molecules compete on roughly equal terms for the binding (=catalytic) site on the enzyme.

Non-competitive inhibitors can bind either to the active site, or to other parts of the enzyme far away from the substrate-binding site. Moreover, non-competitive inhibitors bind to the enzyme-substrate (ES) complex and to the free enzyme. Their binding to this site changes the shape of the enzyme and stops the active site binding substrate(s). Consequently, since there is no direct competition between the substrate and inhibitor for the enzyme, the extent of inhibition depends only on the inhibitor concentration and will not be affected by the substrate concentration.

 

Biological function

Enzymes serve a wide variety of functions inside living organisms. They are indispensable for signal transduction and cell regulation, often via kinases and phosphatases. They also generate movement, with myosin hydrolysing ATP to generate muscle contraction and also moving cargo around the cell as part of the cytoskeleton. Other ATPases in the cell membrane are ion pumps involved in active transport. Enzymes are also involved in more exotic functions, such as luciferase generating light in fireflies.

Viruses can contain enzymes for infecting cells, such as the HIV integrase and reverse transcriptase, or for viral release from cells, like the influenza virus neuraminidase.

An important function of enzymes is in the digestive systems of animals. Enzymes such as amylases and proteases break down large molecules (starch or proteins, respectively) into smaller ones, so they can be absorbed by the intestines. Starch is inabsorbable in the intestine but enzymes hydrolyse the starch chains into smaller molecules such as maltose and eventually glucose, which can then be absorbed. Different enzymes digest different food substances. In ruminants which have a herbivorous diets, bacteria in the gut produce another enzyme, cellulase to break down the cellulose cell walls of plant fiber.

Several enzymes can work together in a specific order, creating metabolic pathways. In a metabolic pathway, one enzyme takes the product of another enzyme as a substrate. After the catalytic reaction, the product is then passed on to another enzyme. Sometimes more than one enzyme can catalyse the same reaction in parallel, this can allow more complex regulation: with for example a low constant activity being provided by one enzyme but an inducible high activity from a second enzyme.

Enzymes determine what steps occur in these pathways. Without enzymes, metabolism would neither progress through the same steps, nor be fast enough to serve the needs of the cell. Indeed, a metabolic pathway such as glycolysis could not exist independently of enzymes. Glucose, for example, can react directly with ATP to become phosphorylated at one or more of its carbons. In the absence of enzymes, this occurs so slowly as to be insignificant. However, if hexokinase is added, these slow reactions continue to take place except that phosphorylation at carbon 6 occurs so rapidly that if the mixture is tested a short time later, glucose-6-phosphate is found to be the only significant product. Consequently, the network of metabolic pathways within each cell depends on the set of functional enzymes that are present.

 

Control activity

There are five main ways that enzyme activity is controlled in the cell.

Regulation of Enzyme Activity

 

While it is clear that enzymes are responsible for the catalysis of almost all biochemical reactions, it is important to also recognize that rarely, if ever, do enzymatic reactions proceed in isolation. The most common scenario is that enzymes catalyze individual steps of multi-step metabolic pathways, as is the case with glycolysis, gluconeogenesis or the synthesis of fatty acids. As a consequence of these lock- step sequences of reactions, any given enzyme is dependent on the activity of preceding reaction steps for its substrate.

In humans, substrate concentration is dependent on food supply and is not usually a physiologically important mechanism for the routine regulation of enzyme activity. Enzyme concentration, by contrast, is continually modulated in response to physiological needs. Three principal mechanisms are known to regulate the concentration of active enzyme in tissues:

 

1. Regulation of gene expression controls the quantity and rate of enzyme synthesis.

 

2. Proteolytic enzyme activity determines the rate of enzyme degradation.

3. Covalent modification of preexisting pools of inactive proenzymes produces active enzymes.

 

Enzyme synthesis and proteolytic degradation are comparatively slow mechanisms for regulating enzyme concentration, with response times of hours, days or even weeks. Proenzyme activation is a more rapid method of increasing enzyme activity but, as a regulatory mechanism, it has the disadvantage of not being a reversible process. Proenzymes are generally synthesized in abundance, stored in secretory granules and covalently activated upon release from their storage sites. Examples of important proenzymes include pepsinogen, trypsinogen and chymotrypsinogen, which give rise to the proteolytic digestive enzymes. Likewise, many of the proteins involved in the cascade of chemical reactions responsible for blood clotting are synthesized as proenzymes. Other important proteins, such as peptide hormones and collagen, are also derived by covalent modification of precursors.

 

Another mechanism of regulating enzyme activity is to sequester enzymes in compartments where access to their substrates is limited. For example, the proteolysis of cell proteins and glycolipids by enzymes responsible for their degradation is controlled by sequestering these enzymes within the lysosome.

In contrast to regulatory mechanisms that alter enzyme concentration, there is an important group of regulatory mechanisms that do not affect enzyme concentration, are reversible and rapid in action, and actually carry out most of the moment- to- moment physiological regulation of enzyme activity. These mechanisms include allosteric regulation, regulation by reversible covalent modification and regulation by control proteins such as calmodulin. Reversible covalent modification is a major mechanism for the rapid and transient regulation of enzyme activity. The best examples, again, come from studies on the regulation of glycogen metabolism where phosphorylation of glycogen synthase and glycogen phosphorylase kinase results in the stimulation of glycogen degradation while glycogen synthesis is coordinately inhibited. Numerous other enzymes of intermediary metabolism are affected by phosphorylation, either positively or negatively. These covalent phosphorylations can be reversed by a separate sub-subclass of enzymes known as phosphatases. Recent research has indicated that the aberrant phosphorylation of growth factor and hormone receptors, as well as of proteins that regulate cell division, often leads to unregulated cell growth or cancer. The usual sites for phosphate addition to proteins are the serine, threonine and tyrosine R group hydroxyl residues.

Enzyme production (transcription and translation of enzyme genes) can be enhanced or diminished by a cell in response to changes in the cell's environment. This form of gene regulation is called enzyme induction and inhibition. For example, bacteria may become resistant to antibiotics such as penicillin because enzymes called beta-lactamases are induced that hydrolyse the crucial beta-lactam ring within the penicillin molecule. Another example are enzymes in the liver called cytochrome P450 oxidases, which are important in drug metabolism. Induction or inhibition of these enzymes can cause drug interactions.

Enzymes can be compartmentalized, with different metabolic pathways occurring in different cellular compartments. For example, fatty acids are synthesized by one set of enzymes in the cytosol, endoplasmic reticulum and the Golgi apparatus and used by a different set of enzymes as a source of energy in the mitochondrion, through β-oxidation.

Enzymes can be regulated by inhibitors and activators. For example, the end product(s) of a metabolic pathway are often inhibitors for one of the first enzymes of the pathway (usually the first irreversible step, called committed step), thus regulating the amount of end product made by the pathways. Such a regulatory mechanism is called a negative feedback mechanism, because the amount of the end product produced is regulated by its own concentration. Negative feedback mechanism can effectively adjust the rate of synthesis of intermediate metabolites according to the demands of the cells. This helps allocate materials and energy economically, and prevents the manufacture of excess end products. Like other homeostatic devices, the control of enzymatic action helps to maintain a stable internal environment in living organisms.

Enzymes can be regulated through post-translational modification. This can include phosphorylation, myristoylation and glycosylation. For example, in the response to insulin, the phosphorylation of multiple enzymes, including glycogen synthase, helps control the synthesis or degradation of glycogen and allows the cell to respond to changes in blood sugar. Another example of post-translational modification is the cleavage of the polypeptide chain. Chymotrypsin, a digestive protease, is produced in inactive form as chymotrypsinogen in the pancreas and transported in this form to the stomach where it is activated. This stops the enzyme from digesting the pancreas or other tissues before it enters the gut. This type of inactive precursor to an enzyme is known as a zymogen.

Some enzymes may become activated when localized to a different environment (eg. from a reducing (cytoplasm) to an oxidising (periplasm) environment, high pH to low pH etc). For example, hemagglutinin of the influenza virus undergoes a conformational change once it encounters the acidic environment of the host cell vesicle causing its activation.

 

Involvement in disease Phenylalanine hydroxylase. Created from PDB 1KW0Since the tight control of enzyme activity is essential for homeostasis, any malfunction (mutation, overproduction, underproduction or deletion) of a single critical enzyme can lead to a genetic disease. The importance of enzymes is shown by the fact that a lethal illness can be caused by the malfunction of just one type of enzyme out of the thousands of types present in our bodies.

One example is the most common type of phenylketonuria. A mutation of a single amino acid in the enzyme phenylalanine hydroxylase, which catalyzes the first step in the degradation of phenylalanine, results in build-up of phenylalanine and related products. This can lead to mental retardation if the disease is untreated.

Another example is when germline mutations in genes coding for DNA repair enzymes cause hereditary cancer syndromes such as xeroderma pigmentosum. Defects in these enzymes cause cancer since the body is less able to repair mutations in the genome. This causes a slow accumulation of mutations and results in the development of many types of cancer in the sufferer.

 

Naming conventions

An enzyme's name is often derived from its substrate or the chemical reaction it catalyzes, with the word ending in -ase. Examples are lactase, alcohol dehydrogenase and DNA polymerase. This may result in different enzymes, called isoenzymes, with the same function having the same basic name. Isoenzymes have a different amino acid sequence and might be distinguished by their optimal pH, kinetic properties or immunologically. Furthermore, the normal physiological reaction an enzyme catalyzes may not be the same as under artifical conditions. This can result in the same enzyme being identified with two different names. E.g. Glucose isomerase, used industrially to convert glucose into the sweetener fructose, is a xylose isomerase in vivo.

Allosteric Enzymes

Allosteric modulation

Allosteric enzymes change their structure in response to binding of effectors. Modulation can be direct, where the effector binds directly to binding sites in the enzyme, or indirect, where the effector binds to other proteins or protein subunits that interact with the allosteric enzyme and thus influence catalytic activity.

In addition to simple enzymes that interact only with substrates and inhibitors, there is a class of enzymes that bind small, physiologically important molecules and modulate activity in ways other than those described above. These are known as allosteric enzymes; the small regulatory molecules to which they bind are known as effectors. Allosteric effectors bring about catalytic modification by binding to the enzyme at distinct allosteric sites, well removed from the catalytic site, and causing conformational changes that are transmitted through the bulk of the protein to the catalytically active site(s).

The hallmark of effectors is that when they bind to enzymes, they alter the catalytic properties of an enzyme's active site. Those that increase catalytic activity are known as positive effectors. Effectors that reduce or inhibit catalytic activity are negative effectors.

Most allosteric enzymes are oligomeric (consisting of multiple subunits); generally they are located at or near branch points in metabolic pathways, where they are influential in directing substrates along one or another of the available metabolic paths. The effectors that modulate the activity of these allosteric enzymes are of two types. Those activating and inhibiting effectors that bind at allosteric sites are called heterotropic effectors. (Thus there exist both positive and negative heterotropic effectors.) These effectors can assume a vast diversity of chemical forms, ranging from simple inorganic molecules to complex nucleotides such as cyclic adenosine monophosphate (cAMP). Their single defining feature is that they are not identical to the substrate.

In many cases the substrate itself induces distant allosteric effects when it binds to the catalytic site. Substrates acting as effectors are said to be homotropic effectors. When the substrate is the effector, it can act as such, either by binding to the substrate-binding site, or to an allosteric effector site. When the substrate binds to the catalytic site it transmits an activity-modulating effect to other subunits of the molecule. Often used as the model of a homotropic effector is hemoglobin, although it is not a branch-point enzyme and thus does not fit the definition on all counts.

There are two ways that enzymatic activity can be altered by effectors: the Vmax can be increased or decreased, or the Km can be raised or lowered. Enzymes whose Km is altered by effectors are said to be K-type enzymes and the effector a K-type effector. If Vmax is altered, the enzyme and effector are said to be V-type. Many allosteric enzymes respond to multiple effectors with V-type and K-type behavior. Here again, hemoglobin is often used as a model to study allosteric interactions, although it is not strictly an enzyme.

In the preceding discussion we assumed that allosteric sites and catalytic sites were homogeneously present on every subunit of an allosteric enzyme. While this is often the case, there is another class of allosteric enzymes that are comprised of separate catalytic and regulatory subunits. The archetype of this class of enzymes is cAMP-dependent protein kinase (PKA), whose mechanism of activation is illustrated in the Figure below. The enzyme is tetrameric, containing two catalytic subunits and two regulatory subunits, and enzymatically inactive. When intracellular cAMP levels rise, one molecule of cAMP binds to each regulatory subunit, causing the tetramer to dissociate into one regulatory dimer and two catalytic monomers. In the dissociated form, the catalytic subunits are fully active; they catalyze the phosphorylation of a number of other enzymes, such as those involved in regulating glycogen metabolism. The regulatory subunits have no catalytic activity.

Representative pathway for the activation of cAMP-dependent protein kinase (PKA). In this example glucagon binds to its' cell-surface receptor, thereby activating the receptor. Activation of the receptor is coupled to the activation of a receptor-coupled G-protein (GTP-binding and hydrolyzing protein) composed of 3 subunits. Upon activation the a-subunit dissociates and binds to and activates adenylate cyclase. Adenylate cylcase then converts ATP to cyclic-AMP (cAMP). The cAMP thus produced then binds to the regulatory subunits of PKA leading to dissociation of the associated catalytic subunits. The catalytic subunits are inactive until dissociated from the regulatory subunits. Once released the catalytic subunits of PKA phosphorylate numerous substrate using ATP as the phosphate donor.

The 19th-century physiologist Claude Bernard enunciated the conceptual basis for metabolic regulation. He observed that living organisms respond in ways that are both quantitatively and temporally appropriate to permit them to survive the multiple challenges posed by changes in their external and internal environments. Walter Cannon subsequently coined the term homeostasis to describe the ability of animals to maintain a constant intracellular environment despite changes in their external environment. We now know that organisms respond to changes in their external and internal environment by balanced, coordinated changes in the rates of specific metabolic reactions. Many human diseases, including cancer, diabetes, cystic fibrosis, and Alzheimers disease, are characterized by regulatory dysfunctions triggered by pathogenic agents or genetic mutations. For example, many oncogenic viruses elaborate protein-tyrosine kinases that modify the regulatory events which control patterns of gene expression, contributing to the initiation and progression of cancer. The toxin from Vibrio cholerae, the causative agent of cholera, disables sensor-response pathways in intestinal epithelial cells by ADP-ribosylating the GTP-binding proteins (G-proteins) that link cell surface receptors to adenylyl cyclase. The consequent activation of the cyclase triggers the flow of water into the intestines, resulting in massive diarrhea and dehydration. Yersinia pestis, the causative agent of plague, elaborates a protein-tyrosine phosphatase that hydrolyzes phosphoryl groups on key cytoskeletal proteins. Knowledge of factors that control the rates of enzyme-catalyzed reactions thus is essential to an

understanding of the molecular basis of disease.

Factors Affecting Enzyme Action

The activity of enzymes is strongly affected by changes in pH and temperature. Each enzyme works best at a certain pH (left graph) and temperature (right graph), its activity decreasing at values above and below that point. This is not surprising considering the importance of

                     tertiary structure (i.e. shape) in enzyme function and noncovalent forces, e.g., ionic interactions and hydrogen bonds, in determining that shape.

Examples:

                     the protease pepsin works best as a pH of 1-2 (found in the stomach) while

                     the protease trypsin is inactive at such a low pH but very active at a pH of 8 (found in the small intestine as the bicarbonate of the pancreatic fluid neutralizes the arriving stomach contents).

Changes in pH alter the state of ionization of charged amino acids (e.g., Asp, Lys) that may play a crucial role in substrate binding and/or the catalytic action itself. Without the unionized -COOH group of Glu-35 and the ionized -COO- of Asp-52, the catalytic action of lysozyme would cease.

Hydrogen bonds are easily disrupted by increasing temperature. This, in turn, may disrupt the shape of the enzyme so that its affinity for its substrate diminishes. The ascending portion of the temperature curve (red arrow in right-hand graph above) reflects the general effect of increasing temperature on the rate of chemical reactions (graph at left). The descending portion of the curve above (blue arrow) reflects the loss of catalytic activity as the enzyme molecules become denatured at high temperatures.

Precursor Activation

The accumulation of a substance within a cell may specifically activate (blue arrow) an enzyme that sets in motion a sequence of reactions for which that substance is the initial substrate. This reduces the concentration of the initial substrate.

In the case if feedback inhibition and precursor activation, the activity of the enzyme is being regulated by a molecule which is not its substrate. In these cases, the regulator molecule binds to the enzyme at a different site than the one to which the substrate binds. When the regulator binds to its site, it alters the shape of the enzyme so that its activity is changed. This is called an allosteric effect.

                     In feedback inhibition, the allosteric effect lowers the affinity of the enzyme for its substrate.

                     In precursor activation, the regulator molecule increases the affinity of the enzyme in the series for its substrate.

Regulation of Enzyme Synthesis

The four mechanisms described above regulate the activity of enzymes already present within the cell.

What about enzymes that are not needed or are needed but not present?

Here, too, control mechanisms are at work that regulate the rate at which new enzymes are synthesized. Most of these controls work by turning on - or off - the transcription of genes.

If, for example, ample quantities of an amino acid are already available to the cell from its extracellular fluid, synthesis of the enzymes that would enable the cell to produce that amino acid for itself is shut down.

Conversely, if a new substrate is made available to the cell, it may induce the synthesis of the enzymes needed to cope with it. Yeast cells, for example, do not ordinarily metabolize lactose and no lactase can be detected in them. However, if grown in a medium containing lactose, they soon begin synthesizing lactase - by transcribing and translating the necessary gene(s) - and so can begin to metabolize the sugar.

E. coli also has a mechanism which regulates enzyme synthesis by controlling translation of a needed messenger RNA.

The Role of Ezymes in Biological Reactions

The laws of thermodymatics apply to chemical reactions anywhere in the universe. No reactions can violate the rules of thermodymatics. All reaction must proceed to a level of minimum energy and maximum entropy or have a favorable balance between the two. Enzyme simply increase the rate (rate is equal to the number of reactant molecules converted to product per unit of time) at which spontaneous reactions take place. Enzymes cannot make a reaction occur that would not proceed spontaneously without the enzyme. The same principle apply to reversible reaction. Enzymes do not alter the equilibrium point of reversible reaction.

Most biological reactions would take place to slow without enzyme for life to exist. The rates would be essentially zero at biological temperatures. For example the oxidation of glucose to CO2 and H2O is spontaneous and proceeds almost completely in the direction stated. However, without enzymes, glucose oxidation occurs do slowly at physiological temperatures that the rate is essentially unmeasureable.

The increases in rate achieve by enzymes, depending on the enzyme and reaction, range from a minimum of about a million to as much as a trillion times faster than the uncatalyzed reactions at equivalent concentrations and temperatures.

Enzyme and Activation Energy

Activation energy is the energy barrier over which the molecules in a system must be raised for a reaction to take place (Figure 1).

http://ntri.tamuk.edu/cell/chapter10/activation.gif

This condition is analogous to a rock resting in a depression at the top of a hill. As long as the rock remains undisturbed, it will not spontaneously roll downhill unless activation energy is applied to the rock. Spontaneous movement over the barrier occurs because molecules, unlike the rock are in constant motion at temperatures above absolute zero. Although the average amount of movement, or kinetic energy, is below the amount required for activation, some molecular collision may raise a number of molecules to the energy level required for the reaction to proceed. The higher the activating barrier, the fewer the molecules that will proceed over the energy barrier per unit time.


Factors Affecting Enzyme Activity

A number of external factors affect the activity of enzymes in speeding conversion of reactants to products. These factors, including variations in the concentration for substrate molecules, temperature, and pH, speed or slow enzymatic activity in highly characteristic patterns.

Lock and key

Substrate Concentration

Enzymes react distinctively to alteration in the concentration of reacting molecules. At very low substrate concentration, collisions between enzyme and substrate molecules are infrequent and reaction proceeds slowly. As the substrate concentration increases, there reaction rate initially increases proportionately as collisions between enzyme molecules and reactants become more frequent Figure 3. When the enzymes begin to approach the maximum rate at which they can combine with reactants and release products, the effects of increasing substrate concentration diminish. At the point at which the enzymes are cycling as rapidly as possible, further increases in substrate concentration have no effect on there reaction rate. At this point the enzyme is saturated and the reaction remains at the saturation level.

Concentration and rate

If the reaction reaches a point at which further increases in reactants have no effect in increasing the rate of the reaction, then there is a good chance that the reaction is catalyzed by an enzyme. Uncatalyzed reactions, in contrast, increase the rate rate of the reaction almost indefinitely as the concentration of reactants increases.

The fact that enzymes combine briefly with their reactants makes them susceptible to inhibition by unreactive molecules that resemble the substrate. The inhibiting molecules can combine with the active site of the enzyme but tend to remain bound without change, blocking access by the normal substrate. As a result, the rate of there reaction slows. If the concentration of the inhibitor becomes high enough, the reaction may stop completely. Inhibition of this type is called competitive because the inhibitor competes with the normal substrate for binding to the active site.
Some inhibitors interfere with enzyme-catalyzed reactions by combining with enzymes at locations outside the active site. These inhibitors, rather than reducing accessibility of the active site to the substrate, cause changes in folding conformation that reduce the ability of the enzyme to lower the activation energy. Because such inhibitors do no directly compete for binding to the active site, their pattern of inhibition is called noncompetitive. Some poisons or toxins exert their damaging effects by acting as enzyme inhibitors. For example, the action of cyanide and carbon monoxide as poisons depends on their ability to inhibit enzyme important the utilization of oxygen in cellular respiration. Poisons and toxins typically act irreversibly by combining so strongly with enzymes, either covalently or nocovalently, that the inhibition is essentially permanent. Some irreversible poisons, rather than combining with the enzyme, destroy enzyme activity by chemically modifying critical amino acid side groups.

Allosteric Inhibition

The cell has built in mechanisms to control directly both enzyme concentration and activity. First cells are able to regulate whether an enzyme is present at all. This type of control regulates protein synthesis and will be discussed in a later chapter. Cells also have ways to control the level of activity of enzymes that have already been synthesized and are present in the cell.

In noncompetitive inhibition, a molecule binds to an enzyme but not at the active site. The other binding site is called the allosteric site (allo - other and steric -structure or space). The molecule that binds to the allosteric site is an inhibitor because it causes a change in the 3-dimensional structure of the enzyme that prevents the substrate from binding to the active site. In cells inhibition usually reversible; that is the inhibitor isn't permanently bound to the enzyme. Irreversible inhibition of enzymes also occurs, due to the presence of a poison. For example, penicillin cause the death of bacteria due to irreversible inhibition of an enzyme needed to form the bacterial cell wall. In humans, hydrogen cyanide irreversibly bind to a very important enzyme (cytochrome oxidase) present in all cells, and this accounts for its lethal effect on the body.

The activity of almost every enzyme is a cell is regulated by feedback inhibition. Feedback inhibition is an example of common biological control mechanism called negative feedback. Just as high temperature will cause furnace to shut off, in a similar manner the product of an enzyme can inhibit a enzyme reaction. When the product is in abundance, it binds competitively with its enzyme's active site; as the product is used up, inhibition is reduced and more product can be produced. In this way the concentration of the product is always controlled within a certain range.

Most enzymatic pathways are also regulated by feedback inhibition, but in these cases the end product of the pathway binds at an allosteric site on the first enzyme of the pathway. This binding shuts down the pathway, and not more product id produced. The reaction series converting theronine to isoleucine is a classic example of allosteric regulation. Five enzymes acting in sequence catalyze the pathway. The final product of the sequence, isoleucine, acts as an inhibitor of the first enzyme of the pathway, threonine deaminase. As the pathway produces isoleucine, any molecules made in excess of cell requirements combine reversibly with threonine deaminase at a location outside the active site. The combination converts threonine deaminase to the T state and inhibits its ability to combine with threonine. The pathway is then turned off. If the concentration of isoleucine later falls as a result of its use in cell synthesis, isoleucine releases from the threonine deaminase enzymes, converting them to the R state in which they have high affinity of the substrate, conversion of threonine to isoleucine takes place.

 

Activation of an allosteric enzyme by an activator is another form of feedback inhibition. Combination of the activator and the allosteric site cause a conformational change in the active site permitting substrate binding and the reaction will be caltalyzed.

Enzyme Regulation Feedback inhibition

 

REACTIONS AND ENZYMES

Energy releasing processes, ones that "generate" energy, are termed exergonic reactions. Reactions that require energy to initiate the reaction are known as endergonic reactions. All natural processes tend to proceed in such a direction that the disorder or randomness of the universe increases (the second law of thermodynamics).

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/exergonic.gif

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/activationener.gif

Time-energy graphs of an exergonic reaction (top) and endergonic reaction (bottom). Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Oxidation/Reduction

Biochemical reactions in living organisms are essentially energy transfers. Often they occur together, "linked", in what are referred to as oxidation/reduction reactions. Reduction is the gain of an electron. Sometimes we also have H ions along for the ride, so reduction also becomes the gain of H. Oxidation is the loss of an electron (or hydrogen). In oxidation/reduction reactions, one chemical is oxidized, and its electrons are passed (like a hot potato) to another (reduced, then) chemical. Such coupled reactions are referred to as redox reactions. The metabolic processes glycolysis, Kreb's Cycle, and Electron Transport Phosphorylation involve the transfer of electrons (at varying energy states) by redox reactions.

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/redox.gif

Passage of electrons from compound A to compound B. When A loses its electrons it is oxidized; when B gains the electrons it is reduced.

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/redoxdiag.gif

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/redoxNAD.gif

Oxidation/reduction via an intermediary (energy carrier) compound, in this case NAD+. Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Anabolism is the total series of chemical reactions involved in synthesis of organic compounds. Autotrophs must be able to manufacture (synthesize) all the organic compounds they need. Heterotrophs can obtain some of their compounds in their diet (along with their energy). For example humans can synthesize 12 of the 20 amino acids, we must obtain the other 8 in our diet. Catabolism is the series of chemical reactions that breakdown larger molecules. Energy is released this way, some of it can be utilized for anabolism. Products of catabolism can be reassembled by anabolic processes into new anabolic molecules.

Enzymes allow many chemical reactions to occur within the homeostasis constraints of a living system. Enzymes function as organic catalysts. A catalyst is a chemical involved in, but not changed by, a chemical reaction. Many enzymes function by lowering the activation energy of reactions. By bringing the reactants closer together, chemical bonds may be weakened and reactions will proceed faster than without the catalyst.

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/catreact.gif

The use of enzymes can lower the activation energy of a reaction (Ea). Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Enzymes can act rapidly, as in the case of carbonic anhydrase (enzymes typically end in the -ase suffix), which causes the chemicals to react 107 times faster than without the enzyme present. Carbonic anhydrase speeds up the transfer of carbon dioxide from cells to the blood. There are over 2000 known enzymes, each of which is involved with one specific chemical reaction. Enzymes are substrate specific. The enzyme peptidase (which breaks peptide bonds in proteins) will not work on starch (which is broken down by human-produced amylase in the mouth).

Enzymes are proteins. The functioning of the enzyme is determined by the shape of the protein. The arrangement of molecules on the enzyme produces an area known as the active site within which the specific substrate(s) will "fit". It recognizes, confines and orients the substrate in a particular direction.

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/indfit.gif

Space filling model of an enzyme working on glucose. Note the shape change in the enzyme (indicated by the red arrows) after glucose has fit into the binding or active site.

The induced fit hypothesis suggests that the binding of the substrate to the enzyme alters the structure of the enzyme, placing some strain on the substrate and further facilitating the reaction. Cofactors are nonproteins essential for enzyme activity. Ions such as K+ and Ca+2 are cofactors. Coenzymes are nonprotein organic molecules bound to enzymes near the active site. NAD (nicotinamide adenine dinucleotide).

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/enzsub.gif

A cartoonish view of the formation of an enzyme-substrate complex. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Enzymatic pathways form as a result of the common occurrence of a series of dependent chemical reactions. In one example, the end product depends on the successful completion of five reactions, each mediated by a specific enzyme. The enzymes in a series can be located adjacent to each other (in an organelle or in the membrane of an organelle), thus speeding the reaction process. Also, intermediate products tend not to accumulate, making the process more efficient. By removing intermediates (and by inference end products) from the reactive pathway, equilibrium (the tendency of reactions to reverse when concentrations of the products build up to a certain level) effects are minimized, since equilibrium is not attained, and so the reactions will proceed in the "preferred" direction.

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/metabolfbk.gif

Negative feedback and a metabolic pathway. The production of the end product (G) in sufficient quantity to fill the square feedback slot in the enzyme will turn off this pathway between step C and D. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Temperature: Increases in temperature will speed up the rate of nonenzyme mediated reactions, and so temperature increase speeds up enzyme mediated reactions, but only to a point. When heated too much, enzymes (since they are proteins dependent on their shape) become denatured. When the temperature drops, the enzyme regains its shape. Thermolabile enzymes, such as those responsible for the color distribution in Siamese cats and color camouflage of the Arctic fox, work better (or work at all) at lower temperatures.

Concentration of substrate and product also control the rate of reaction, providing a biofeedback mechanism.

Activation, as in the case of chymotrypsin, protects a cell from the hazards or damage the enzyme might cause.

Changes in pH will also denature the enzyme by changing the shape of the enzyme. Enzymes are also adapted to operate at a specific pH or pH range.

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/enzpH.gif

Plot of enzyme activity as a function of pH for several enzymes. Note that each enzyme has a range of pH at which it is active as well as an optimal pH at which it is most active. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman, used with permission.

Allosteric Interactions may allow an enzyme to be temporarily inactivated. Binding of an allosteric effector changes the shape of the enzyme, inactivating it while the effector is still bound. Such a mechanism is commonly employed in feedback inhibition. Often one of the products, either an end or near-end product act as an allosteric effector, blocking or shunting the pathway.

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/allostery.gif

Action of an allosteric inhibitor as a negative control on the action of an enzyme.

Competitive Inhibition works by the competition of the regulatory compound and substrate for the binding site. If enough regulatory compound molecules bind to enough enzymes, the pathway is shut down or at least slowed down. PABA, a chemical essential to a bacteria that infects animals, resembles a drug, sulfanilamide, that competes with PABA, shutting down an essential bacterial (but not animal) pathway.

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/reversein1.gif

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/reversein2.gif

Top: general diagram showing competitor in the active site normally occupied by the natural substrate; Bottom: specific case of succinate dehydrogenase and its natural substrate (succinate) and competitors (oxalate et al.).

Noncompetitive Inhibition occurs when the inhibitory chemical, which does not have to resemble the substrate, binds to the enzyme other than at the active site. Lead binds to SH groups in this fashion. Irreversible Inhibition occurs when the chemical either permanently binds to or massively denatures the enzyme so that the tertiary structure cannot be restored. Nerve gas permanently blocks pathways involved in nerve message transmission, resulting in death. Penicillin, the first of the "wonder drug" antibiotics, permanently blocks the pathways certain bacteria use to assemble their cell wall components.

The four mechanisms described above regulate the activity of enzymes already present within the cell.

What about enzymes that are not needed or are needed but not present?

Here, too, control mechanisms are at work that regulate the rate at which new enzymes are synthesized. Most of these controls work by turning on or off the transcription of genes.

If, for example, ample quantities of an amino acid are already available to the cell from its extracellular fluid, synthesis of the enzymes that would enable the cell to produce that amino acid for itself is shut down.

Conversely, if a new substrate is made available to the cell, it may induce the synthesis of the enzymes needed to cope with it. Yeast cells, for example, do not ordinarily metabolize lactose and no lactase can be detected in them. However, if grown in a medium containing lactose, they soon begin synthesizing lactase by transcribing and translating the necessary gene(s) and so can begin to metabolize the sugar.

 

REGULATION OF METABOLITE FLOW CAN BE ACTIVE OR PASSIVE

Enzymes that operate at their maximal rate cannot respond to an increase in substrate concentration, and can respond only to a precipitous decrease in substrate concentration. For most enzymes, therefore, the average intracellular concentration of their substrate tends to be close to the Km value, so that changes in substrate concentration generate corresponding changes in metabolite

flux. Responses to changes in substrate level represent an important but passive means for coordinating metabolite flow and maintaining homeostasis in quiescent cells. However, they offer limited scope for responding to changes in environmental variables. The mechanisms that regulate enzyme activity in an active manner in response to internal and external signals are discussed below.

 

Metabolite Flow Tends to Be Unidirectional

Despite the existence of short-term oscillations in metabolite concentrations and enzyme levels, living cells exist in a dynamic steady state in which the mean

concentrations of metabolic intermediates remain relatively constant over time. While all chemical reactions are to some extent reversible, in living cells the reaction products serve as substrates forand are removed byother enzyme-catalyzed reactions.

 

Many nominally reversible reactions thus occur unidirectionally. This succession of coupled metabolic reactions is accompanied by an overall change in free energy that favors unidirectional metabolite flow. The unidirectional flow of metabolites through a pathway with a large overall negative change in free energy is analogous to the flow of water through a pipe in which one end is lower than the other. Bends or kinks in the pipe simulate individual enzyme-catalyzed steps with a small negative or positive change in free energy. Flow of water through the pipe nevertheless remains unidirectional due to the overall change in height, which corresponds to the overall change in free energy in a pathway.

 

 

 

COMPARTMENTATION ENSURES METABOLIC EFFICIENCY

& SIMPLIFIES REGULATION

 

In eukaryotes, anabolic and catabolic pathways that interconvert common products may take place in specific subcellular compartments. For example, many of the enzymes that degrade proteins and polysaccharides reside inside organelles called lysosomes. Similarly, fatty acid biosynthesis occurs in the cytosol, whereas fatty acid oxidation takes place within mitochondria.

Segregation of certain metabolic pathways within specialized cell types can provide further physical compartmentation. Alternatively, possession of

one or more unique intermediates can permit apparently opposing pathways to coexist even in the absence of physical barriers. For example, despite many shared intermediates and enzymes, both glycolysis and gluconeogenesis

are favored energetically. This cannot be true if all the reactions were the same. If one pathway was favored energetically, the other would be accompanied by

a change in free energy G equal in magnitude but opposite in sign. Simultaneous spontaneity of both pathways results from substitution of one or more reactions

by different reactions favored thermodynamically in the opposite direction. The glycolytic enzyme phosphofructokinase is replaced by the gluconeogenic enzyme fructose-1,6-bisphosphatase. The ability of enzymes to discriminate between the structurally similar coenzymes NAD+ and NADP+ also results in a form of compartmentation, since it segregates the electrons of NADH that are destined for ATP generation from those of NADPH that participate in the reductive steps in many biosynthetic pathways.

 

Controlling an Enzyme That Catalyzes a Rate-Limiting Reaction Regulates

an Entire Metabolic Pathway

While the flux of metabolites through metabolic pathways involves catalysis by numerous enzymes, active control of homeostasis is achieved by regulation of only a small number of enzymes. The ideal enzyme for regulatory intervention is one whose quantity or catalytic efficiency dictates that the reaction it catalyzes is slow relative to all others in the pathway. Decreasing the catalytic efficiency or the quantity of the catalyst for the bottleneck or rate-limiting reaction immediately reduces metabolite flux through the entire pathway. Conversely, an increase in either its quantity or catalytic efficiency enhances flux through the pathway as a whole.

For example, acetyl-CoA carboxylase catalyzes the synthesis of malonyl-CoA, the first committed reaction of fatty acid biosynthesis. When synthesis of

malonyl-CoA is inhibited, subsequent reactions of fatty acid synthesis cease due to lack of substrates. Enzymes that catalyze rate-limiting steps serve as natural governors of metabolic flux. Thus, they constitute efficient targets for regulatory intervention by drugs. For example, inhibition by statin drugs of HMG-CoA reductase, which catalyzes the rate-limiting reaction of cholesterogenesis, curtails synthesis of cholesterol.

 

REGULATION OF ENZYME QUANTITY

The catalytic capacity of the rate-limiting reaction in a metabolic pathway is the product of the concentration of enzyme molecules and their intrinsic catalytic efficiency. It therefore follows that catalytic capacity can be influenced both by changing the quantity of enzyme present and by altering its intrinsic catalytic efficiency.

Control of Enzyme Synthesis

Enzymes whose concentrations remain essentially constant over time are termed constitutive enzymes. By contrast, the concentrations of many other enzymes depend upon the presence of inducers, typically substrates or structurally related compounds, that initiate their synthesis. Escherichia coli grown on glucose will, for example, only catabolize lactose after addition of a β-galactoside, an inducer that initiates synthesis of a β-galactosidase and a galactoside permease. Inducible enzymes of humans include tryptophan pyrrolase, threonine dehydrase, tyrosine-α-ketoglutarate aminotransferase, enzymes of the urea cycle, HMG-CoA reductase, and cytochrome P450. Conversely, an excess of a metabolite may curtail synthesis of its cognate

enzyme via repression. Both induction and repression involve cis elements, specific DNA sequences located upstream of regulated genes, and trans-acting regulatory proteins.

Control of Enzyme Degradation

The absolute quantity of an enzyme reflects the net balance between enzyme synthesis and enzyme degradation, where ks and kdeg represent the rate constants for the overall processes of synthesis and degradation, respectively.

Changes in both the ks and kdeg of specific enzymes occur in human subjects.

Protein turnover represents the net result of enzyme synthesis and degradation. By measuring the rates of incorporation of 15N-labeled amino acids into protein

and the rates of loss of 15N from protein, Schoenheimer deduced that body proteins are in a state of dynamic equilibrium in which they are continuously

synthesized and degraded. Mammalian proteins are degraded both by ATP and ubiquitin-dependent pathways and by ATP-independent pathways.

Susceptibility to proteolytic degradation can be influenced by the presence of ligands such as substrates, coenzymes, or metal ions that alter protein conformation. Intracellular ligands thus can influence the rates at which specific enzymes are degraded.

Enzyme levels in mammalian tissues respond to a wide range of physiologic, hormonal, or dietary factors. For example, glucocorticoids increase the concentration of tyrosine aminotransferase by stimulating ks, and glucagondespite its antagonistic physiologic effects increases ks fourfold to fivefold. Regulation of liver arginase can involve changes either in ks or in kdeg. After

a protein-rich meal, liver arginase levels rise and arginine synthesis decreases. Arginase levels also rise in starvation, but here arginase degradation decreases,

whereas ks remains unchanged. Similarly, injection of glucocorticoids and ingestion of tryptophan both elevate levels of tryptophan oxygenase. While the

hormone raises ks for oxygenase synthesis, tryptophan specifically lowers kdeg by stabilizing the oxygenase against proteolytic digestion.

 

MULTIPLE OPTIONS ARE AVAILABLE FOR REGULATING CATALYTIC ACTIVITY

In humans, the induction of protein synthesis is a complex multistep process that typically requires hours to produce significant changes in overall enzyme level. By contrast, changes in intrinsic catalytic efficiency effected by binding of dissociable ligands (allosteric regulation) or by covalent modification achieve regulation of enzymic activity within seconds. Changes in protein level serve long-term adaptive requirements, whereas changes in catalytic efficiency are best suited for rapid and transient alterations in metabolite flux.

ALLOSTERIC EFFECTORS REGULATE CERTAIN ENZYMES

Feedback inhibition refers to inhibition of an enzyme in a biosynthetic pathway by an end product of that pathway. For example, for the biosynthesis of D from A catalyzed by enzymes Enz1 through Enz3, high concentrations of D inhibit conversion of A to B. Inhibition results not from the backing up of intermediates but from the ability of D to bind to and inhibit Enz1. Typically, D binds at an allosteric site spatially distinct from the catalytic site of the target

enzyme. Feedback inhibitors thus are allosteric effectors and typically bear little or no structural similarity to the substrates of the enzymes they inhibit. In this example, the feedback inhibitor D acts as a negative allosteric effector of Enz1.

IS NOT SYNONYMOUS WITH FEEDBACK INHIBITION

In both mammalian and bacterial cells, end products feed back and control their own synthesis, in many instances by feedback inhibition of an early biosynthetic enzyme. We must, however, distinguish between feedback regulation, a phenomenologic term devoid of mechanistic implications, and feedback inhibition, a mechanism for regulation of enzyme activity. For example, while dietary cholesterol decreases hepatic synthesis of cholesterol, this feedback regulation does not involve feedback inhibition. HMG-CoA reductase, the rate-limiting enzyme of cholesterologenesis, is affected,

but cholesterol does not feedback-inhibit its activity.

Regulation in response to dietary cholesterol involves curtailment by cholesterol or a cholesterol metabolite of the expression of the gene that encodes HMG-CoA reductase (enzyme repression).

MANY HORMONES ACT THROUGH ALLOSTERIC SECOND MESSENGERS

Nerve impulsesand binding of hormones to cell surface receptorselicit changes in the rate of enzymecatalyzed reactions within target cells by inducing the release or synthesis of specialized allosteric effectors called second messengers. The primary or first messenger is the hormone molecule or nerve impulse. Second messengers include 3′,5′-cAMP, synthesized from ATP by

the enzyme adenylyl cyclase in response to the hormone epinephrine, and Ca2+, which is stored inside the endoplasmic reticulum of most cells. Membrane depolarization resulting from a nerve impulse opens a membrane channel that releases calcium ion into the cytoplasm, where it binds to and activates enzymes involved in the regulation of contraction and the mobilization of stored

glucose from glycogen. Glucose then supplies the increased energy demands of muscle contraction. Other second messengers include 3′,5′-cGMP and polyphosphoinositols, produced by the hydrolysis of inositol phospholipids by hormone-regulated phospholipases.

 

REGULATORY COVALENT MODIFICATIONS CAN BE REVERSIBLE OR IRREVERSIBLE

In mammalian cells, the two most common forms of covalent modification are partial proteolysis and phosphorylation. Because cells lack the ability to reunite the two portions of a protein produced by hydrolysis of a peptide bond, proteolysis constitutes an irreversible modification. By contrast, phosphorylation is a reversible modification process. The phosphorylation of proteins on seryl, threonyl, or tyrosyl residues, catalyzed by protein kinases, is thermodynamically spontaneous. Equally spontaneous is the hydrolytic removal of these phosphoryl groups by enzymes called protein phosphatases.

 

PROTEASES MAY BE SECRETED AS CATALYTICALLY INACTIVE PROENZYMES

Certain proteins are synthesized and secreted as inactive precursor proteins known as proproteins. The proproteins of enzymes are termed proenzymes or zymogens. Selective proteolysis converts a proprotein by one or more successive proteolytic clips to a form that exhibits the characteristic activity of the mature protein, eg, its enzymatic activity. Proteins synthesized as proproteins include the hormone insulin (proprotein = proinsulin), the digestive enzymes pepsin, trypsin, and chymotrypsin (proproteins = pepsinogen, trypsinogen, and chymotrypsinogen, respectively), several factors of the blood clotting and blood clot dissolution cascades , and the connective tissue protein collagen (proprotein = procollagen).

 

Proenzymes Facilitate Rapid Mobilization of an Activity in Response

to Physiologic Demand

The synthesis and secretion of proteases as catalytically inactive proenzymes protects the tissue of origin (eg, the pancreas) from autodigestion, such as can occur in pancreatitis. Certain physiologic processes such as digestion are intermittent but fairly regular and predictable. Others such as blood clot formation, clot dissolution, and tissue repair are brought on line only in

response to pressing physiologic or pathophysiologic need. The processes of blood clot formation and dissolution clearly must be temporally coordinated to

achieve homeostasis. Enzymes needed intermittently but rapidly often are secreted in an initially inactive form since the secretion process or new synthesis of the required proteins might be insufficiently rapid for response to a pressing pathophysiologic demand such as the loss of blood.

 

Activation of Prochymotrypsin Requires Selective Proteolysis

Selective proteolysis involves one or more highly specific proteolytic clips that may or may not be accompanied by separation of the resulting peptides. Most importantly, selective proteolysis often results in conformational changes that create the catalytic site of an enzyme. Note that while His 57 and Asp 102 reside on the B peptide of α-chymotrypsin, Ser 195 resides on the C peptide. The conformational changes that accompany selective proteolysis of prochymotrypsin (chymotrypsinogen) align the three residues of the charge-relay network, creating the catalytic site. Note also that contact and catalytic residues can be located on different peptide chains but still be within bond-forming distance of bound substrate.

 

 

REVERSIBLE COVALENT MODIFICATION REGULATES KEY MAMMALIAN ENZYMES

Mammalian proteins are the targets of a wide range of covalent modification processes. Modifications such as glycosylation, hydroxylation, and fatty acid acylation introduce new structural features into newly synthesized proteins that tend to persist for the lifetime of the protein. Among the covalent modifications that regulate protein function (eg, methylation, adenylylation), the most common by far is phosphorylation-dephosphorylation.

Protein kinases phosphorylate proteins by catalyzing transfer of the terminal phosphoryl group of ATP to the hydroxyl groups of seryl, threonyl, or tyrosyl

residues, forming O-phosphoseryl, O-phosphothreonyl, or O-phosphotyrosyl residues, respectively. Some protein kinases target the side chains of histidyl, lysyl, arginyl, and aspartyl residues. The unmodified form of the protein can be regenerated by hydrolytic removal of phosphoryl groups, catalyzed by protein phosphatases.

A typical mammalian cell possesses over 1000 phosphorylated

proteins and several hundred protein kinases and protein phosphatases that catalyze their interconversion. The ease of interconversion of enzymes between

their phospho- and dephospho- forms in part dephosphorylation as a mechanism for regulatory control. Phosphorylation-dephosphorylation permits the functional properties of the affected enzyme to be altered only for as long as it serves a specific need. Once the need has passed, the enzyme can be converted back to its original form, poised to respond to the next stimulatory event. A second factor underlying the widespread use of protein phosphorylation-dephosphorylation lies in the chemical properties of the phosphoryl group itself.

In order to alter an enzymes functional properties, any modification of its chemical structure must influence the proteins three-dimensional configuration.

The high charge density of protein-bound phosphoryl groupsgenerally −2 at physiologic pHand their propensity to form salt bridges with arginyl residues

make them potent agents for modifying protein structure and function. Phosphorylation generally targets amino acids distant from the catalytic site itself. Consequent conformational changes then influence an enzymes

intrinsic catalytic efficiency or other properties. In this sense, the sites of phosphorylation and other covalent modifications can be considered another form of allosteric site. However, in this case the allosteric ligand binds covalently to the protein.

 

PROTEIN PHOSPHORYLATION IS EXTREMELY VERSATILE

Protein phosphorylation-dephosphorylation is a highly versatile and selective process. Not all proteins are subject to phosphorylation, and of the many hydroxyl groups on a proteins surface, only one or a small subset are targeted. While the most common enzyme function affected is the proteins catalytic efficiency, phosphorylation can also alter the affinity for substrates, location

within the cell, or responsiveness to regulation by allosteric ligands. Phosphorylation can increase an enzymes catalytic efficiency, converting it to its active form in one protein, while phosphorylation of another converts it into an intrinsically inefficient, or inactive, form.

 

Many proteins can be phosphorylated at multiple sites or are subject to regulation both by phosphorylation-dephosphorylation and by the binding of allosteric ligands. Phosphorylation-dephosphorylation at any one site can be catalyzed by multiple protein kinases or protein phosphatases. Many protein kinases and most protein phosphatases act on more than one protein and are

themselves interconverted between active and inactive forms by the binding of second messengers or by covalent modification by phosphorylation-dephosphorylation. The interplay between protein kinases and protein phosphatases, between the functional consequences of phosphorylation at different sites, or between phosphorylation sites and allosteric sites provides the basis for regulatory networks that integrate multiple environmental input signals to evoke an appropriate coordinated cellular response. In these sophisticated regulatory networks, individual enzymes respond to different environmental signals. For example, if an enzyme can be phosphorylated at a single site by more than one protein kinase, it can be converted from a catalytically efficient to an inefficient (inactive) form, or vice versa, in response to any one of several signals. If the protein kinase is activated in response to a signal different from the signal that activates the protein phosphatase, the phosphoprotein becomes a decision node. The functional output, generally catalytic activity, reflects the phosphorylation state. This state or degree of phosphorylation is determined by the relative activities of the protein kinase and protein phosphatase, a reflection of the presence and relative strength of the environmental signals that act through each. The ability of many protein kinases and protein phosphatases to target more than one protein provides a means for an environmental signal to coordinately regulate multiple metabolic processes. For example, the enzymes 3-hydroxy- 3-methylglutaryl-CoA reductase and acetyl-CoA carboxylasethe rate-controlling enzymes for cholesterol and fatty acid biosynthesis, respectivelyare phosphorylated and inactivated by the AMP-activated protein kinase. When this protein kinase is activated either through phosphorylation by yet another protein kinase or in response to the binding of its allosteric activator 5′-AMP, the two major pathways responsible for the synthesis of lipids from acetyl-CoA both are inhibited. Interconvertible enzymes and the enzymes responsible for their interconversion do not act as mere on and off switches working independently of one another.

 

 

 

Enzymes are biological catalysts responsible for supporting almost all of the chemical reactions that maintain animal homeostasis. Because of their role in maintaining life processes, the assay and pharmacological regulation of enzymes have become key elements in clinical diagnosis and therapeutics. The macromolecular components of almost all enzymes are composed of protein, except for a class of RNA modifying catalysts known as ribozymes. Ribozymes are molecules of ribonucleic acid that catalyze reactions on the phosphodiester bond of other RNAs.

Enzymes are found in all tissues and fluids of the body. Intracellular enzymes catalyze the reactions of metabolic pathways. Plasma membrane enzymes regulate catalysis within cells in response to extracellular signals, and enzymes of the circulatory system are responsible for regulating the clotting of blood. Almost every significant life process is dependent on enzyme activity.

http://www.youtube.com/watch?v=Ofs0mfkl370

 

Enzyme Classifications

 

Traditionally, enzymes were simply assigned names by the investigator who discovered the enzyme. As knowledge expanded, systems of enzyme classification became more comprehensive and complex. Currently enzymes are grouped into six functional classes by the International Union of Biochemists (I.U.B.).


 

 

Number

Classification

Biochemical Properties

1.

Oxidoreductases

Act on many chemical groupings to add or remove hydrogen atoms.

2.

Transferases

Transfer functional groups between donor and acceptor molecules. Kinases are specialized transferases that regulate metabolism by transferring phosphate from ATP to other molecules.

3.

Hydrolases

Add water across a bond, hydrolyzing it.

4.

Lyases

Add water, ammonia or carbon dioxide across double bonds, or remove these elements to produce double bonds.

5.

Isomerases

Carry out many kinds of isomerization: L to D isomerizations, mutase reactions (shifts of chemical groups) and others.

6.

Ligases

Catalyze reactions in which two chemical groups are joined (or ligated) with the use of energy from ATP.

 

These rules give each enzyme a unique number. The I.U.B. system also specifies a textual name for each enzyme. The enzyme's name is comprised of the names of the substrate(s), the product(s) and the enzyme's functional class. Because many enzymes, such as alcohol dehydrogenase, are widely known in the scientific community by their common names, the change to I.U.B.-approved nomenclature has been slow. In everyday usage, most enzymes are still called by their common name.

 

Enzymes are also classified on the basis of their composition. Enzymes composed wholly of protein are known as simple enzymes in contrast to complex enzymes, which are composed of protein plus a relatively small organic molecule. Complex enzymes are also known as holoenzymes. In this terminology the protein component is known as the apoenzyme, while the non-protein component is known as the coenzyme or prosthetic group where prosthetic group describes a complex in which the small organic molecule is bound to the apoenzyme by covalent bonds; when the binding between the apoenzyme and non-protein components is non-covalent, the small organic molecule is called a coenzyme. Many prosthetic groups and coenzymes are water-soluble derivatives of vitamins. It should be noted that the main clinical symptoms of dietary vitamin insufficiency generally arise from the malfunction of enzymes, which lack sufficient cofactors derived from vitamins to maintain homeostasis.

 

The non-protein component of an enzyme may be as simple as a metal ion or as complex as a small non-protein organic molecule. Enzymes that require a metal in their composition are known as metalloenzymes if they bind and retain their metal atom(s) under all conditions with very high affinity. Those which have a lower affinity for metal ion, but still require the metal ion for activity, are known as metal-activated enzymes.

 

Enzymes in the Diagnosis of Pathology

http://www.youtube.com/watch?v=5hrU6_tic7s&feature=related

 

The measurement of the serum levels of numerous enzymes has been shown to be of diagnostic significance. This is because the presence of these enzymes in the serum indicates that tissue or cellular damage has occurred resulting in the release of intracellular components into the blood. Hence, when a physician indicates that he/she is going to assay for liver enzymes, the purpose is to ascertain the potential for liver cell damage. Commonly assayed enzymes are the amino transferases: alanine transaminase, ALT (sometimes still referred to as serum glutamate-pyruvate aminotransferase, SGPT) and aspartate aminotransferase, AST (also referred to as serum glutamate-oxaloacetate aminotransferase, SGOT); lactate dehydrogenase, LDH; creatine kinase, CK (also called creatine phosphokinase, CPK); gamma-glutamyl transpeptidase, GGT. Other enzymes are assayed under a variety of different clinical situations but they will not be covered here.

 

The typical liver enzymes measured are AST and ALT. ALT is particularly diagnostic of liver involvement as this enzyme is found predominantly in hepatocytes. When assaying for both ALT and AST the ratio of the level of these two enzymes can also be diagnostic. Normally in liver disease or damage that is not of viral origin the ratio of ALT/AST is less than 1. However, with viral hepatitis the ALT/AST ratio will be greater than 1. Measurement of AST is useful not only for liver involvement but also for heart disease or damage. The level of AST elevation in the serum is directly proportional to the number of cells involved as well as on the time following injury that the AST assay was performed. Following injury, levels of AST rise within 8 hours and peak 24-36 hours later. Within 3-7 days the level of AST should return to pre-injury levels, provided a continuous insult is not present or further injury occurs. Although measurement of AST is not, in and of itself, diagnostic for myocardial infarction, taken together with LDH and CK measurements (see below) the level of AST is useful for timing of the infarct.

 

The measurement of LDH is especially diagnostic for myocardial infarction because this enzyme exist in 5 closely related, but slightly different forms (isozymes). The 5 types and their normal distribution and levels in non-disease/injury are listed below.

 

LDH 1 - Found in heart and red-blood cells and is 17% - 27% of the normal serum total.

LDH 2 - Found in heart and red-blood cells and is 27% - 37% of the normal serum total.

LDH 3 - Found in a variety of organs and is 18% - 25% of the normal serum total.

LDH 4 - Found in a variety of organs and is 3% - 8% of the normal serum total.

LDH 5 - Found in liver and skeletal muscle and is 0% - 5% of the normal serum total.

 

Following a myocardial infarct the serum levels of LDH rise within 24-48 hours reaching a peak by 2-3 days and return to normal in 5-10 days. Especially diagnostic is a comparison of the LDH-1/LDH-2 ratio. Normally, this ration is less than 1. A reversal of this ration is referred to as a "flipped LDH.". Following an acute myocardial infart the flipped LDH ratio will appear in 12-24 hours and is definitely present by 48 hours in over 80% of patients. Also important is the fact that persons suffering chest pain due to angina only will not likely have altered LDH levels.

 

CPK is found primarily in heart and skeletal muscle as well as the brain. Therefore, measurement of serum CPK levels is a good diagnostic for injury to these tissues. The levels of CPK will rise within 6 hours of injury and peak by around 18 hours. If the injury is not persistent the level of CK returns to normal within 2-3 days. Like LDH, there are tissue-specific isozymes of CPK and there designations are described below.

CPK3 (CPK-MM) is the predominant isozyme in muscle and is 100% of the normal serum total.

CPK2 (CPK-MB) accounts for about 35% of the CPK activity in cardiac muscle, but less than 5% in skeletal muscle and is 0% of the normal serum total.

CPK1 (CPK-BB) is the characteristic isozyme in brain and is in significant amounts in smooth muscle and is 0% of the normal serum total.

 

Since most of the released CPK after a myocardial infarction is CPK-MB, an increased ratio of CPK-MB to total CPK may help in diagnosis of an acute infarction, but an increase of total CPK in itself may not. CPK-MB levels rise 3-6 hours after a myocardial infarct and peak 12-24 hours later if no further damage occurs and returns to normal 12-48 hours after the infarct.

 

CLINICAL ENZYMOLOGY

http://www.youtube.com/watch?v=5hrU6_tic7s&feature=related

PLASMA ENZYMES


Measurements of the activity of enzymes in plasma are of value in the diagnosis and management of a wide variety of diseases. Most enzymes measured in plasma are primarily intracellular, being released into the blood when there is damage to cell membranes, but many enzymes, for example renin, complement factors and coagulation factors, are actively secreted into the blood, where they fulfil their physiological functions. Small amounts of intracellular enzymes are present in the blood as a result of normal cell turnover. When damage to cells occurs, increased amounts of enzymes will be released and their concentrations in the blood will rise. However, such increases are not always due to tissue damage. Other possible causes include:

increased cell turnover

cellular proliferation (e.g. neoplasia)

increased enzyme synthesis (enzyme induction)

obstruction to secretion

decreased clearance.

Little is known about the mechanisms by which enzymes are removed from the circulation. Small molecules, such as amylase, are filtered by the glomeruli but most enzymes are probably removed by reticuloendothelial cells. Plasma amylase activity rises in acute renal failure but, in general, changes in clearance rates are not known to be important as causes of changes in plasma enzyme levels.

Plasma contains many functional enzymes, which a actively secreted into plasma. For example, enzymes blood coagulation. On the other hand, there are a few non functional enzymes in plasma, which are coming out from cells of various tissues due to normal wear and tear. Their normal levels in blood are very low; but are drastically increased during cell death (necrosis) or disease. Therefore assays of these enzymes are very useful in diagnosis diseases.

Enzyme assays usually depend on the measurement the catalytic activity of the enzyme, rather than the concentration of the enzyme protein itself. Since each enzyme molecule can catalyze the reaction of many molecules of substrate, measurement of activity provides great sensitivity. It is, however, important that the conditions of the assay are optimized and standardized to give reliable and reproducible results. Reference ranges for plasma enzymes are dependent on assay conditions, for example temperature, and may also be subject to physiological influences. It is thus important to be aware of both the reference range for the laboratory providing the assay and the physiological circumstances when interpreting the results of enzyme assays.

One international unit is the amount of enzyme that will convert one micromole of substrate per minute per litre of sample and is abbreviated as U/L. The SI Unit (System Internationale) expression is more scientific, where or Katal (catalytic activity) is defined as the number of mole of substrate transformed per second per litre of sample. Katal is abbreviated as kat or k (60 U = 1 μkat and 1 nk = 0.06 U).

Disadvantages of enzyme assays

A major disadvantage in the use of enzymes for the diagnosis of tissue damage is their lack of specificity to a particular tissue or cell type. Many enzymes are common to more than one tissue, with the result that an increase in the plasma activity of a particular enzyme could reflect damage to any one of these tissues. This problem may be obviated to some extent in two ways:

first, different tissues may contain (and thus release when they are damaged) two or more enzymes in different proportions; thus alanine and aspartate aminotransferases are both present in cardiac and skeletal muscle and hepatocytes, but there is only a very little alanine aminotransferase in either type of muscle;

second, some enzymes exist in different forms (isoforms), colloquially termed isoenzymes (although, strictly, the term 'isoenzyme' refers only to a genetically determined isoform). Individual isoforms are often characteristic of a particular tissue: although they may have similar catalytic activities, they often differ in some other measurable property, such as heat stability or sensitivity to inhibitors.

After a single insult to a tissue, the activity of intracellular enzymes in the plasma rises as they are released from the damaged cells, and then falls as the enzymes are cleared. It is thus important to consider the time at which the blood sample is taken in relation to the insult. If taken too soon, there may have been insufficient time for the enzyme to reach the blood- stream and if too late, it may have been completely cleared. As with all diagnostic techniques, data acquired from measurements of enzymes in plasma must always be assessed in the light of whatever clinical and other information is available, and their limitations borne in mind.

 

LACTATE DEHYDROGENASE (LDH) (LD)

The total LDH is generally tested by reaction of the serum sample with pyruvate and NADH2. LDH will convert pyruvate to lactate, and in turn NADH is use up by the reaction.

Normal value of LDH in serum is 100-200 U/L. Values the upper range are generally seen in children. Strenuous exercise will slightly increase the value. LDH level is 100 times more inside the RBC than in plasma, and therefore minor amount of hemolysis will result in a false-positive test.

LDH and Heart Attack

In myocardial infarction, total LDH activity is increased, while H4 iso-enzyme is increased 5-10 times more.

Differential diagnosis: Increase in total LDH level is seen in hemolytic anemias, hepatocellular damage, muscular dystrophy, carcinomas, leukemias, and any condition which causes necrosis of body cells. Since total LDH is increased in many conditions, the study of isozymes of LDH is of great importance.

Isoenzymes of LDH

 

LDH enzyme is a tetramer with four subunits. But the subunit may be either H (heart) or M (muscle) polypeptide chains. These two are the products of two different genes.

Although both of them have the same molecular weight (32 kD), there are minor amino acid variations. So five combinations of H and M chains are possible; H4, H3M, H2M2, M3H and M4 varieties, forming five iso-enzymes.

 

 

 

All these five forms are seen in all persons. M4 form is seen in skeletal muscles; it is not inhibited by pyruvate. But H4 form is seen in heart and is inhibited by pyruvate. Normally LDH-2 (H3M1) concentration in blood is greater than LDH-1 (H4); but this pattern is reversed in myocardial infarction; this is called flipped pattern. The iso-enzymes are usual ly separated by cellulose acetate electrophoresis at pH 8.6. They are then identified by adding the reactants finally producing a colour reaction. . Lactate dehydrogenase isoenzymes (as percentage of total):

LDH1 14-26 %

LDH2 29-39 %

LDH3 20-26 %

LDH4 8-16%

LDH5 6-16 %

 

CREATINE KINASE(CK)

http://www.youtube.com/watch?v=6r5Ddlcq26s

 

Creatine → Creatine phosphate

It was called as creatine phosphokinase in old literature.

Normal serum value for CK is 15-100 U/L for males and 10-80 U/L for females.

CK and Heart Attack CK value in serum is increased in myocardial infarction. The CK level starts to rise within three hours of infarction. Therefore, CK estimation is very useful to detect early cases, where ECG changes may be ambiguous. The CK level is not increased in hemolysis or in congestive cardiac failure; and therefore CK has an advantage over LDH.

CK and Muscle Diseases he level of CK in serum is very much elevated in muscular dystrophies (500 -1500 IU/L). The level is very high in the early phases of the disease. In such patients a fall in CK level is indicative of deteriorating condition, because by that time, all muscle mass is destroyed. In female carriers of this X-linked disease (genotypically heterozygous), CK is seen to be moderately raised. CK level is highly elevated in crush injury, fracture and acute cerebrovascular accidents. Estimation of total CK is employed in muscular dystrophies and MB iso-enzyme is estimated in myocardial infarction.

Iso-enzymes of CK CK is a dimer; each subunit has a molecular weight of 40,000. The subunits are called B for brain and M for muscle. They are products of loci in chromosomes 14 and 19 respectively. Therefore three iso-enzymes are seen in circulation. Normally CK2 is only 5% of the total activity. Even doubling the value in CK2 (MB) iso-enzyme may not be detected, if total value of CK alone is estimated. Hence the detection of MB-iso-enzyme is important in myocardial infarction. CK-MB < 6 % of total CK in normal conditions.

The above three iso-enzymes are cytosolic. A fourth variety, called CK-mt is located in mitochondria and constitutes about 15% of total CK activity. Its gene is located in chromosome 15. CK1 may be complexed with immunoglobulin; and then termed macroCK. CK1-lgG causes false-positive diagnosis of myocardial infarction because it has an electrophoretic mobility close to CK2.

For quantitating MB iso-enzyme, anti-MM antiserum is added to the patient's serum. This will precipitate MM iso-enzyme. The supernatant serum is used for the CK estimation. Here it is assumed that BB isoenzyme is negligible in quantity, which is correct if there is no brain disease. CK iso-enzymes can also be identified by electrophoresis.

ASPARTATE AMINO TRANSFERASE (AST)

It is also called as serum glutamate-oxaloacetate transaminase (SGOT). AST needs pyridoxal phosphate as co-enzyme. AST is estimated by taking aspartate, α-ketoglutarate, pyridoxal phosphate (vitamin B6) and patient' serum as the source of AST. The oxaloacetate formed may be allowed to react with dinitrophenyl hydrazine to produce a colour which is estimated colorimetrically at 520 nm.

Normal serum level of AST is 8-40 U/L or (0,1-0,45 mmol/(hour´L))

It is significantly elevated in myocardial infarction. It if moderately elevated in liver diseases. However, a marked increase in AST may be seen in primary hepatoma. AST has two iso-enzymes; cytoplasmic and mitochondrial. In mile degree of tissue injury, cytoplasmic form is seen in serum. Mitochondrial type is seen in severe injury.

ALANINE AMINO TRANSFERASE (ALT)

It is also called as serum glutamate-pyruvate transaminase (SGPT). ALT needs pyridoxal phosphate as co-enzyme.

Normal serum level of ALT is 5-30 U/L or (0,1-0,68 mmol/(hour´L))

Very high values (100 to 1000 U/L) are seen in acute hepatitis, either toxic or viral in origin. Both ALT and AST are increased in liver diseases, but ALT >AST. Moderate increase (25 to 100 U/L) may be seen in chronic liver disease such as cirrhosis, and malignancy in liver. A sudden fall in ALT level in cases of hepatitis is a very bad prognostic sign.

Ritis coefficient (AST/ALT) in normal conditions is 1,330,42.

http://www.youtube.com/watch?v=nXRWkorYFXc

 

10007

 

10009

 

Post mortem specimen of cirrhotic liver and enlarged spleen

 

 

Alkaline phosphatase (ALP)

It is a non- specific enzyme which hydrolyses aliphatic, aromatic or heterocyclic compounds. The pH optimum for the enzyme reaction is between 9 and 10. It is prodused by osteoblasts of bone, and localized in cell memmbranes (ecto-enzyme).

Normal serum level of ALP is 40-125 U/L or 0,5-1,3 mmol/(hour´ L).

In children the upper level of normal value may be more, becouse of the increased osteoblastic activity. Mild increase is noticed during pregnancy, due to production of placental isoenzyme.

Moderate (2-3 times) increase in ALP level is seen in hepatic diseases such as hepatitis, alcoholic hepatosis or hepatocellular carcinoma. Very high levels of ALP (10-12 times of upper limit) may be noticed in extrahepatic obstructions or cholestasis. ALP is produced by epithelial cells of biliary canaliculi and obstruction of bile with consequent irritation of epithelial cells leads to secretion of ALP into serum.

10011

Drastically high levels of ALP (10-25 times of upper limit) are also seen in bone diseases where osteoblastic activity is enhanced such as Paget's disease, rickets, osteomalacia, osteoblastoma, metastatic carcinoma of bone and hyperparathyroidism (Paget's disease or osteitis deformans was described in 1877 by Sir James Paget).

Iso-enzymes of Alkaline Phosphatase

1. α-1 ALP moves in α -1 position, it is synthesised by epithelial cells of biliary canaliculi. It is about 10% of total activity and is increased in obstructive jaundice and to some extent in metastatic carcinoma of liver.

2. α -2 heat labile ALP is stable at 56C; but loses its activity when kept at 65C for 30 minutes. It is produced by hepatic cells. Therefore absence of α -1 with exaggerated α -2 band suggests hepatitis. This liver iso-enzyme forms about 25% of total ALP.

3. α -2 heat stable ALP will not be destroyed at 65 C but is inhibited by phenylalanine. It is of placental origin, which is found in blood in normal pregnancy. An isoenzyme closely resembling the placental form is characteristically seen in circulation in about 15% cases of carcinoma of lung, liver and gut and named as Regan isoenzyme (after the first patient in whom it was detected or carcinoplacental iso-enzyme. Chronic heavy smoking also increases Regan iso-enzyme level in blood. Normal level is only 1 % of the total ALP.

4. Pre-ß ALP is of bone origin and elevated levels are seen in bone diseases. This is the most heat labile (destroyed at 56C, 10 min). Wheat germ lectin will precipitate bone iso-enzyme. This constitutes about 50% of normal ALP activity.

5. γ-ALP is inhibited by phenylalanine and originates from intestinal cells. It is increased in ulcerative colitis. About 10% of plasma ALP are of intestinal variety.

6. The leucocyte alkaline phosphatase (LAP) is significantly decreased in chronic myeloid leukemia. It is increased in lymphomas.

ALP has different isoforms. Although ALP is a monomer, depending on the number of sialic acid residues, the charged groups differ. Such different forms are detected in agar gel electrophoresis.

 

NUCLEOTIDE PHOSPHATASE (NTP)

It is also known as 5' nucleotidase. This enzyme hydrolyses 5' nucleotides to corresponding nucleosides at an optimum pH of 7.5. It is a marker enzyme for plasma membranes and is seen as an ecto-enzyme (enzyme present on the cell membrane).

Usually, AMP is used as substrate, which is hydrolysed to adenosine and inorganic phosphate. The latter reacts with ammonium molybdate to produce the yellow ammonium phosphomolybdate, which is estimated colorimetrically. However, ALP will also catalyse the same reaction. Serum samples contain both ALP and NTP. These are distinguished by Nickel ions which inhibit NTP but not ALP.

Normal NTP level in serum is 2-10 U/L. It is moderately increased in hepatitis and highly elevated in biliary obstruction. Unlike ALP, the level is unrelated with osteoblastic activity and therefore is unaffected by bone diseases.

 

GAMMA GLUTAMYL TRANSFERASE (GGT)

The old name was gamma glutamyl transpeptidase. It can transfer γ-glutamyl residues to substrate. In the body it is used in the synthesis of glutathione. GGT has 11 iso-enzymes. It is seen in liver, kidney, pancreas, intestinal cells and prostate gland.

Normal serum value of GGT is 6-45 U/L in male and 5-30 U/L in female. It is slightly higher in normal males, due to the presence of prostate gland. This value is moderately increased in infective hepatitis and prostate cancers. The GGT level is highly elevated in alcoholism, obstructive jaundice and neoplasm's of liver. GGT-2 is positive for 90% of hepatocellular carcinomas. It is not elevated in cardiac or skeletal diseases.

GGT is a microsomal enzyme. Its activity is induced by alcohol, phenobarbitone and rifampicin. GGT is clinically important because of its sensitivity to detect alcohol abuse. GGT is increased in alcoholics even when other liver function tests are within normal limits. GGT level is rapidly decreased within a few days when the person stops to take alcohol. Increase in GGT level is generally proportional to the amount of alcohol intake.

 

ACID PHOSPHATASE (ACP)

It hydrolyses phosphoric acid ester at pH between 4 and 6. Methods for assay are the same as described for ALP; but the pH of the medium is kept at 5 to 5.4.

Normal serum value for ACP is 2.5-12 U/L or 0,025-0,12 mmol/(hour´ L).

ACP is secreted by prostate cells, RBC, platelets and WBC. Isoenzymes of ACP are described. Erythrocyte ACP gene is located in chromosome 2; osteoclast ACP gene is on chromosome 19; lysosomal gene is on 11 and prostate ACP gene is on 13. The prostate iso-enzyme is inactivated by tartaric acid. Cupric ions inhibit erythrocyte ACP. Normal level of tartrate labile fraction of ACP is 1 U/L.

ACP total value is increased in prostate cancer and highly elevated in bone metastasis of prostate cancer. In these conditions, the tartrate labile iso-enzyme is elevated. This assay is very helpful in follow up of treatment of prostate cancers. ACP is therefore an important tumour marker.

Since blood cells contain excess quantity of ACP, must be taken to prevent hemolysis while taking blood from the patient. Prostate massage may also increase to value. So blood may be collected for ACP estimation before per rectal examination of patient. ACP is present in high concentration in semen, a finding which is used in forensic medicine in investigation of rape.

 

PROSTATE SPECIFIC ANTIGEN (PSA)

It is produced from the secretory epithelium of prostal gland. It is normally secreted into seminal fluid, where it is necessary for the liquefaction of seminal coagulum. It is a serine protease, and is a 32 kD glycoprotein; encoded in chromosome number 19. In blood it is bound to alpha-2 macroglobulin and alpha-1-antitrypsin; a very smal fraction is in the free from also.

Normal value is 1 -5 g/L. It is very specific for prostate activity. Values between 4-10 g/L is seen in benign prostate enlargement; but values above 10 g/L is indicative of prostate cancer.

 

CHOLINESTERASE (ChE)

Acetyl cholinesterase or true ChE or Type 1 ChE can act mainly on acetyl choline. It is present in nerve endings and in RBCs. About 25 allelic forms are reported. Normal serum range is 2-12 U/ml. Newly formed RBC will contain good quantity of ChE which is slowly reduced according to the age of the cell. Therefore, ChE level in RBCs will be proportional to the reticulocyte count. Organophosphorus insecticides (Parathione) irreversibly inhibit ChE in RBCs. Measurement of ChE level in RBCs is useful to determine the amount of exposure in persons working with these insecticides.

Pseudocholinesterase or type II ChE is non-specific and can hydrolyse acyl esters. It is produced mainly by liver cells. Normal serum level is 8-18 U/ml. Succinyl choline is a widely used as muscle relaxant. It is a structural analogue of ACh, and so competitively fix on post-synaptic receptors of ACh. Succinyl choline is hydrolysed by the liver ChE within 2-4 minutes. But in certain persons the ChE activity may be absent; this is a genetically transmitted condition. In such individuals when succinyl choline is given during surgery, it may take hours to get the drug metabolised. Very prolonged scoline apnoea may result in 'nightmare of anaesthetist'. The pseudocholinesterase level in serum is reduced in viral hepatitis, cirrhosis, hepatocellular carcinoma, metastatic cancer of liver and in malnutrition.

http://www.youtube.com/watch?v=-gIqZ8IxctE

 

GLUCOSE-6-PHOSPHATE DEHYDROGENASE

GPD is a dimer with identical subunits. This is an important enzyme in the hexose monophosphate shunt pathway of glucose. It is mainly used for production of NADPH . It has a special role in the RBC metabolism. Due to the presence of oxygen, hydrogen peroxide is continuously formed inside the RBC. Peroxide will destroy biomembranes, and RBCs are lysed. Normal value of GPD in RBC is 125-250 U/1012 cells. Nearly 400 variants (isoforms) of GPD are described.

 

AMYLASE

This splits starch to maltose. It is activated by calcium, chloride and fluoride ions. There are 18 phenotypes. It is produced by pancreas and salivary glands; they are products of different genes located in chromosome 1.

http://www.youtube.com/watch?v=AEsQxzeAry8

Normal serum value is 50-120 U/L, (12-32 g/(hour× L)).

The value is increased about 1000 times in acute pancreatitis which is a life-threatening condition. The peak values are seen between 5-12 hours after the onset of disease and returns to normal levels within 2-4 days after the acute phase has subsided. Moderate increase in serum levels are seen in chronic pancreatitis, mumps (parotitis), obstruction of pancreatic duct and in renal disease. In the last condition, the enzyme is not excreted through urine properly and hence serum value is raised. Normal urine value is 20-160 g/(hour× L) or (less than 375 U/L). It is increased in acute pancreatitis. It is increased on the 1 st day and remains to be elevated for 7-10 days.

 

LIPASE

It will hydrolyse triglyceride to β-monoglyceride and fatty acid. Molecular weight is 54,000. The gene is in chromosome 10. The enzyme is present in pancreatic secretion. Normal serum range is 0.2-1.5 U/L. It is highly elevated in acute pancreatitis and this persists for 7-14 days. Thus, lipase remains elevated longer than amylase. Moreover, lipase is not increased in mumps. Therefore, lipase estimation has advantage over amylase. It is moderately increased in carcinoma of pancreas, biliary diseases and perforating peptic ulcers.

 

ALDOLASE (ALD)

It is a tetrameric enzyme with A and B subunits; so there are 5 iso-enzymes. It is a glycolytic enzyme. Normal range of serum is 1.5-7 U/L. It is drastically elevated in muscle damages such as progressive muscular dystrophy, poliomyelitis, myasthenia gravis and multiple sclerosis. It is a very sensitive early index in muscle wasting diseases.

 

ENOLASE

It is a glycolytic enzyme. Neuron-specific enolase (NSE) is an iso-enzyme seen in neural tissues and Apudomas. NSE is a tumour marker for cancers associated with neuro-endocrine origin, small cell lung cancer, neuroblastoma, pheochromocytoma, medullary carcinoma of thyroid, etc. It is measured by RIA or ELISA. Upper limit of NSE is 12 μg/ml.

 

Usage enzymes in medical practice

 

Enzymo-pathologies disoders of enzymes action. Enzymo-pathologies takes place actually at any disease (its so called secondary enzymo-pathologies). But there also are more than one thousand different forms of congenital molecular pathologies (primary enzymo-pathologies). Mutations (genetic disorders) were found to be the causes of these kind of enzymo-pathologies.

The examples of primary enzymo-pathologies:

1) Enzyme phenylalanine 4-monooxygenase is absent in about 1 in every 10000 human beings owing to a recessive mutant gene. In the absence of this enzyme, a pathway of phenylalanine breakdown and tyrosine is not formed. In

Alloenzyme - Isoenzyme

 

another pathway phenylalanine undergoes transamination with alpha-ketoglutarate to yield phenylpyruvic acid, which accumulates in the blood and is excreted in the urine. In childhood excess circulating phenylpyruvate impairs normal brain development, causing severe mental retardation. This condition, phenylketonuria, was among the first genetic defects of metabolism recognized in man. Restriction of dietary phenylalanine during childhpood prevents the mental retardation.

2) The absence of homogentisic acid 1,2-dioxygenase causes urinary excretion of phenylpyruvic and homogentisic acids. The urinary of people genetically defective in homogentisic acid 1,2-dioxygenase contains homogentisic acid, which when made alkaline and exposed to oxygen, turns dark because it is oxidized and polimerized to a black melanin pigment. This condition is known as alkaptonuria. Patients with this condition have abnormal pigmentation of the connective tissue.

3) D-galactose is converted into D-glucose in the liver by special reactions which have attracted much attention because they are subject to genetic defects in man, resulting in different forms of the hereditary disease galactosemia. The deffect or absence of enzyme galactose 1-phosphate uridylyltransferase causes the increase of D-galactose in the blood. Galactose 1-phosphate uridylyltransferase is present in normal fetal liver but is lacking in infants with galactosemia. Galactosemic infants suffer from cataract of the lens of the eye as well as mental disorders. This condition can be successfully treated by withholding milk and other sources of galactose from the diet during infancy and childhood.

 

Enzymo-therapy. In digestive tract diseases (deficit of digestive enzymes) pepsin and HCI, enzymes of pancreas (pancreatin, oraza, panzinorm) are recomended to use.

Proteolytic enzymes trypsin and chemotrypsin are used for processing of wounds in burns, ulcers for the a proteolysis and deleting of necrotic tissues.

Trypsin, ribonuclease, DNA-ase are used for the splitting and deleting of fibrin from the pleural cavity, for the dilution and deleting of sputum from the respiratory ways in acute and chronic respiratory diseases, for the thrombophlebitis treatment.

Fibrinolysin, streptokinase are used for the splitting of thrombus.

Hyaluronidase (lidase) is used for the acceleration of different medicines penetration into the biological tissues as well as for the resorption of scars and hematoma.

Cytochrome C is used in the intoxication of CO, H2S and other substances oppressing the tissue respiration.

Glucoso-oxidase is used for the lavage of wounds and burns as antiseptic.

Thrombin is used for preventing and stop of bleeding.

 

The using of coenzymes. ATP is used in heart diseases, muscle dystrophia.

TPP (thiamin pyrophosphate) is used in heart diseases, and nervous system pathology.

FMN (flavinmononucleotide) is used for treatment of skin diseases, keratitis, conjunctivitis.

NAD and NADP are used for improvement of oxidative-reduction processes in organism.

 

 

The using of inhibitors of enzymes. Inhibitors of proteolytic enzymes (kontrical, trasilol) are used in acute pancreatitis for inhibition trypsin, chemotrypsin activity. Irreversible inhibitors

Some enzyme inhibitors react with the enzyme and form a covalent adduct with the protein. The inactivation produced by this type of inhibitor is irreversible. A class of these compounds called suicide inhibitors includes eflornithine a drug used to treat the parasitic disease sleeping sickness. Penicillin and its derivatives also act in this manner. With these drugs, the compound is bound in the active site and the enzyme then converts the inhibitor into an activated form that reacts irreversibly with one or more with amino acid residues.

 

The coenzyme folic acid (left) and the anti-cancer drug methotrexate (right) are very similar in structure. As a result, methotrexate is a competitive inhibitor of many enzymes that use folates.

 

Uses of inhibitors

 

Inhibitors are often used as drugs, but they can also act as poisons. However, the difference between a drug and a poison is usually only a matter of amount, since most drugs are toxic at some level, as Paracelsus wrote, "In all things there is a poison, and there is nothing without a poison." Equally, antibiotics and other anti-infective drugs are just specific poisons that can kill a pathogen but not its host.

 

 

An example of an inhibitor being used as a drug is aspirin, which inhibits the COX-1 and COX-2 enzymes that produce the inflammation messenger prostaglandin, thus suppressing pain and inflammation. The poison cyanide is an irreversible enzyme inhibitor that combines with the copper and iron in the active site of the enzyme cytochrome c oxidase and blocks cellular respiration.

 

In many organisms inhibitors may act as part of a feedback mechanism. If an enzyme produces too much of one substance in the organism, that substance may act as an inhibitor for the enzyme that produces it, causing production of the substance to slow down or stop when there is sufficient amount. This is a form of negative feedback.

 

 

Diacarb inhibitor of carbohydrase. This enzyme regulates the metabolism of Na+ and K+ in kidney canaliculus and diuresis.

Isoenzymes

Another type of regulation of metabolic activity is through the participation of isozymes, multiple forms of a given enzyme that occur within a single species of organism or even in a single cell. Such multiple forms can be detected and separated by gel electrophoresis of cell extracts; since they are coded by different genes, they differ in amino acid composition and thus in their isoelectric pH values.

Lactate dehydrogenase, one of the first enzymes in this class to be studied intensively, occurs as five different isozymes in the tissues of vertebrates. All these isozymes have been isolated. They all catalyze the same overall reaction, Lactate + NAD+ pyruvate + NADH + H+

in which NAD+ and NADH symbolize the oxidized and reduced forms, respectively, of nicotinamide adenine dinucleotide. All five isozymes have the same molecular weight, about 134,000, and all contain four polypeptide chains, each of molecular weight 33,500. The five isozymes consist of five different combinations of two different kinds of polypeptide chains, designated A and B. The isozyme predominating in skeletal muscle has four identical A chains and is designated A4; another, which predominates in heart, has four identical chains and is designated B4. The other three isozymes have the composition 3, A2B2, and 3. Single A and chains have been isolated and found to differ significantly in amino acid content and sequence. When single A and chains, which are inactive, are mixed in appropriate proportions, all the different isozymes of lactate dehydrogenase can be made to form spontaneously in the test tube and all have full catalytic activity.

Genetic research indicates that the amino acid sequences of the two different polypeptide chains A and of lactate dehydrogenase are coded by two different genes. The biosynthesis of the two types of chains and thus the relative amounts of the lactate dehydrogenase isozymes present in a given cell are under genetic regulation. Isozymes of lactate dehydrogenase exist in different proportions in different tissues. Moreover, the relative proportions of the lactate dehydrogenase isozymes in a tissue may change during embryological development. They are also important in diagnosis of heart and liver disease.

Careful kinetic study of the lactate dehydrogenase isozymes has revealed that although they all catalyze the same reaction, they differ in their dependence on substrate concentration, particularly pyruvate, as well as their Vmax values when pyruvate is the substrate. The isozyme A4, characteristic of skeletal muscle and embryonic tissues, reduces pyruvate to lactate at a relatively high rate. The B4 isozyme, characteristic of the heart and other red muscles, reduces pyruvate at a relatively low rate. Moreover, the dehydrogenation of lactate catalyzed by the B4 isozyme is strongly inhibited by pyruvate. The other lactate dehydrogenase isozymes have kinetic properties intermediate between those of the A4 and B4 isozymes, in proportion to their relative content of A and chains.

Isozymes are now known for a great many different enzymes. They usually consist of tightly associated mixtures of different kinds of polypeptide chains, in which are blended the specific kinetic and binding properties contributed by each type of chain. Many allosteric enzymes occur as two or more isozymes that vary in sensitivity to their allosteric modulators. In other cases different isozymes are present in different intracellular compartments.

The study of isozymes has grown into one of fundamental significance in the investigation of the molecular basis of cellular differentiation and morphogenesis. Many proteins in cells, not only those with catalytic activity, may occur in multiple forms.

Enzyme cofactors

Many enzymes require the presence of an additional, nonprotein, cofactor.

                     Some of these are metal ions such as Zn2+ (the cofactor for carbonic anhydrase), Cu2+, Mn2+, K+, and Na+.

                     Some cofactors are small organic molecules called coenzymes. The B vitamins

o                                            thiamine (B1)

o                                            riboflavin (B2) and

o                                            nicotinamide

are precursors of coenzymes.

Coenzymes may be covalently bound to the protein part (called the apoenzyme) of enzymes as a prosthetic group. Others bind more loosely and, in fact, may bind only transiently to the enzyme as it performs its atalytic act.

LysozymeTarget

 

 

 

Lysozyme: a model of enzyme action

A number of lysozymes are found in nature; in human tears and egg white, for examples. The enzyme is antibacterial because it degrades the polysaccharide that is found in the cell walls of many bacteria. It does this by catalyzing the insertion of a water molecule at the position indicated by the red arrow. This hydrolysis breaks the chain at that point.

The bacterial polysaccharide consists of long chains of alternating amino sugars:

                     N-acetylglucosamine (NAG)

                     N-acetylmuramic acid (NAM)

These hexose units resemble glucose except for the presence of the side chains containing amino groups.

Lysozyme is a globular protein with a deep cleft across part of its surface. Six hexoses of the substrate fit into this cleft.

                     With so many oxygen atoms in sugars, as many as 14 hydrogen bonds form between the six amino sugars and certain amino acid R groups such as Arg-114, Asn-37, Asn-44, Trp-62, Trp-63, and Asp-101.

                     Some hydrogen bonds also form with the C=O groups of several peptide bonds.

                     In addition, hydrophobic interactions may help hold the substrate in position.

X-ray crystallography has shown that as lysozyme and its substrate unite, each is slightly deformed. The fourth hexose in the chain (ring #4) becomes twisted out of its normal position. This imposes a strain on the C-O bond on the ring-4 side of the oxygen bridge between rings 4 and 5. It is just at this point that the polysaccharide is broken. A molecule of water is inserted between these two hexoses, which breaks the chain. Here, then, is a structural view of what it means to lower activation energy. The energy needed to break this covalent bond is lower now that the atoms connected by the bond have been distorted from their normal position.

As for lysozyme itself, binding of the substrate induces a small (~0.75Å) movement of certain amino acid residues so the cleft closes slightly over its substrate. So the "lock" as well as the "key" changes shape as the two are brought together. (This is sometimes called "induced fit".)

The amino acid residues in the vicinity of rings 4 and 5 provide a plausible mechanism for completing the catalytic act. Residue 35, glutamic acid (Glu-35), is about 3Å from the -O- bridge that is to be broken. The free carboxyl group of glutamic acid is a hydrogen ion donor and available to transfer H+ to the oxygen atom. This would break the already-strained bond between the oxygen atom and the carbon atom of ring 4.

 

 

Enzymes_pH_temp

 

 

Now having lost an electron, the carbon atom acquires a positive charge. Ionized carbon is normally very unstable, but the attraction of the negatively-charged carboxyl ion of Asp-52 could stabilize it long enough for an -OH ion (from a spontaneously dissociated water molecule) to unite with the carbon. Even at pH 7, water spontaneously dissociates to produce H+ and OH- ions. The hydrogen ion (H+) left over can replace that lost by Glu-35.

In either case, the chain is broken, the two fragments separate from the enzyme, and the enzyme is free to attach to a new location on the bacterial cell wall and continue its work of digesting it.

Regulatory Enzymes

All enzymes exhibit various features that could conceivably be elements in the regulation of their activity in living cells. All have a characteristic optimum pH, which makes possible alteration of their catalytic rates with changes in intracellular pH. The rates of all enzymatic reactions also depend on the substrate concentration, which may vary significantly under intercellular conditions. Moreover, many enzymes require either metal ions, such as Mg2+ or K+, or coenzymes for activity, suggesting that fluctuations in the concentration of these metals or coenzymes in the cell can regulate enzyme activity. However, over and above these properties of all enzymes, some enzymes possess other properties that specifically endow them with regulatory roles in metabolism. Such more highly specialized forms are called reguiatory enzymes. There are two major types of regulatory enzymes: (1) allosteric enzymes, whose catalytic activity is modulated through the noncovalent binding of a specific metabolite at a site on the protein other than the catalytic site, and (2) covalently modulated enzymes, which are interconverted between active and inactive forms by the action of other enzymes. Some of the enzymes in the second class also respond to noncovalent allosteric modulators. These two types of regulatory enzymes are responsive to alterations in the metabolic state of a cell or tissue on a relatively short time scale allosteric enzymes within seconds and covalently regulated enzymes within minutes.

Allosteric Enzymes

In many multienzyme systems the end product of the reaction sequence may act as a specific inhibitor of an enzyme at or near the beginning of the sequence, with the result that the rate of the entire sequence of reactions is determined by the steady-state concentration of the end product. The classical example is the multienzyme sequence catalyzing the conversion of L-threonine to L-isoleucine, which occurs in five enzyme-catalyzed steps. The first enzyme of the sequence, L-threonine dehydratose, is strongly inhibited by L-isoleucine, the end product, but not by any other intermediate in the sequence. The kinetic characteristics of the inhibition by isoleucine are atypical; the inhibition is neither competitive with the substrate L-threonine, nor is it noncompetitive. Isoleucine is quite specific as an inhibitor; other amino acids or related compounds do not inhibit. This type of inhibition is variously called end-product inhibition or feedback inhibition. The first enzyme in this sequence, that which is inhibited by the end product, is called an allosteric enzyme. The term allosteric denotes "another space" or "another structure"; allosteric enzymes possess, in addition to the catalytic site, the "other space," to which the specific effector r modulator is reversibly and noncovalently bound. In general, the allosteric site is as specific for binding the modulator as the catalytic site is for binding the substrate. Some modulators, e.g., L-isoleucine for threonine dehydratase, are inhibitory and therefore called inhibitory or negative modulators. Other allosteric enzymes may have stimulatory, or positive, modulators. When an allosteric enzyme has only one specific modulator, it is said to be monovlent. Some allosteric enzymes respond to two or more specific modulators, each bound to a specific site on the enzyme; they are polyvalent. Moreover, a given allosteric enzyme may have both positive and negative modulators. Two or more multienzyme systems may be connected by one or more polyvalent enzymes in a control network.

The first step in a multienzyme reaction sequence, i.e., the step catalyzed by the allosteric enzyme, is usually irreversible under intracellular conditions. It is often called the committing reaction; once it occurs, all the ensuing reactions of the sequence take place. Clearly, it is good strategy for the cell to regulate a metabolic pathway at its first step, to achieve maximum economy of metabolites.

Allosteric enzymes are usually much larger in molecular weight, more complex, and often more difficult to purify than ordinary enzymes because nearly all known allosteric enzymes are oligomeric and thus have two or more polypeptide chain subunits, usually in an even number; some contain many chains. Allosteric enzymes show a number of anomalous properties. Some are unstable at 0C but stable at room or body temperature, unlike single-chain enzymes, which do not show cold lability.

Allosteric enzymes show two different types of control, heterotropic and homotropic, depending on the nature of the modulating molecule. Heterotropic enzymes are stimulated or inhibited by an effector or modulator molecule other than their substrates. For the heterotropic enzyme threonine dehydratase the substrate is threonine and the modulator is L-isoleucine. In homotropic enzymes, on the other hand, the substrate also functions as the modulator. Homotropic enzymes contain two or more binding sites for the substrate; modulation of these enzymes depends on how many of the substrate sites are occupied. However, a great many (if not most) allosteric enzymes are of mixed homotropic-heterotropic type, in which both the substrate and some other metabolite(s) may function as modulators.

 

http://www.indstate.edu/thcme/mwking/pkacamp.jpg

 

 

Mechanism of the Regulatory Activity of Allosteric Enzymes

Much attention has been focused on the molecular mechanisms by which the binding of the modulator to the regulatory site of an allosteric enzyme can change the activity of the catalytic site. One of the most fruitful avenues of research and speculation has been evoked by the striking functional similarities between allosteric enzymes and hemoglobin.

Two types of models have been proposed for the cooperative interactions of the subunits of hemoglobin - the sequential model and the symmetry model. These models are also applicable to allosteric enzymes having multiple subunits, which are assumed to occur in two different conformations.

According to the symmetry model, binding the first substrate molecule in creases the tendency of the remaining subunits to undergo transition to the high-affinity form through an all-or-none effect, all subunits being either in their low-affinity or their high-affinity forms.

The sequential model also postulates that the catalytic subunits have two conformational states, but it differs from the symmetry model in postulating that the subunits may un-undergo individual sequential changes in conformation; between the all-on and all-off states there may be many intermediate conformational states of the enzyme molecule, each having its own intrinsic catalytic activity.

 

 

Usage enzymes in medical practice

Enzymo-pathologies disoders of enzymes action. Enzymo-pathologies takes place actually at any disease (its so called secondary enzymo-pathologies). But there also are more than one thousand different forms of congenital molecular pathologies (primary enzymo-pathologies). Mutations (genetic disorders) were found to be the causes of these kind of enzymo-pathologies.

The examples of primary enzymo-pathologies:

1) Enzyme phenylalanine 4-monooxygenase is absent in about 1 in every 10000 human beings owing to a recessive mutant gene. In the absence of this enzyme, a pathway of phenylalanine breakdown and tyrosine is not formed. In another pathway phenylalanine undergoes transamination with alpha-ketoglutarate to yield phenylpyruvic acid, which accumulates in the blood and is excreted in the urine. In childhood excess circulating phenylpyruvate impairs normal brain development, causing severe mental retardation. This condition, phenylketonuria, was among the first genetic defects of metabolism recognized in man. Restriction of dietary phenylalanine during childhpood prevents the mental retardation.

http://www.youtube.com/watch?v=hpaki7F4HR0

http://www.youtube.com/watch?v=CWfrVS4Bm1Y&feature=related

 

2) The absence of homogentisic acid 1,2-dioxygenase causes urinary excretion of phenylpyruvic and homogentisic acids. The urinary of people genetically defective in homogentisic acid 1,2-dioxygenase contains homogentisic acid, which when made alkaline and exposed to oxygen, turns dark because it is oxidized and polimerized to a black melanin pigment. This condition is known as alkaptonuria. Patients with this condition have abnormal pigmentation of the connective tissue.

3) D-galactose is converted into D-glucose in the liver by special reactions which have attracted much attention because they are subject to genetic defects in man, resulting in different forms of the hereditary disease galactosemia. The deffect or absence of enzyme galactose 1-phosphate uridylyltransferase causes the increase of D-galactose in the blood. Galactose 1-phosphate uridylyltransferase is present in normal fetal liver but is lacking in infants with galactosemia. Galactosemic infants suffer from cataract of the lens of the eye as well as mental disorders. This condition can be successfully treated by withholding milk and other sources of galactose from the diet during infancy and childhood.

http://www.youtube.com/watch?v=lKaNgfya-DA

 

Enzymo-therapy. In digestive tract diseases (deficit of digestive enzymes) pepsin and HCI, enzymes of pancreas (pancreatin, oraza, panzinorm) are recomended to use.

Proteolytic enzymes trypsin and chemotrypsin are used for processing of wounds in burns, ulcers for the a proteolysis and deleting of necrotic tissues.

Trypsin, ribonuclease, DNA-ase are used for the splitting and deleting of fibrin from the pleural cavity, for the dilution and deleting of sputum from the respiratory ways in acute and chronic respiratory diseases, for the thrombophlebitis treatment.

Fibrinolysin, streptokinase are used for the splitting of thrombus.

Hyaluronidase (lidase) is used for the acceleration of different medicines penetration into the biological tissues as well as for the resorption of scars and hematoma.

Cytochrome C is used in the intoxication of CO, H2S and other substances oppressing the tissue respiration.

Glucoso-oxidase is used for the lavage of wounds and burns as antiseptic.

Thrombin is used for preventing and stop of bleeding.

The using of coenzymes. ATP is used in heart diseases, muscle dystrophia.

TPP (thiamin pyrophosphate) is used in heart diseases, and nervous system pathology.

FMN (flavinmononucleotide) is used for treatment of skin diseases, keratitis, conjunctivitis.

NAD and NADP are used for improvement of oxidative-reduction processes in organism.

The using of inhibitors of enzymes. Inhibitors of proteolytic enzymes (kontrical, trasilol) are used in acute pancreatitis for inhibition trypsin, chemotrypsin activity.

Diacarb inhibitor of carbohydrase. This enzyme regulates the metabolism of Na+ and K+ in kidney canaliculus and diuresis.

 

 

Fat-soluble vitamins

Although fat-soluble vitamins have been studied intensively and widely used in human nutrition, we know less about their specific biological function than about the water-soluble vitamins.

Vitamin A.

Vitamin A occurs in two common forms, vitamin A1, or retinol, the form most common in mammalian tissues and marine fishes, and vitamin, A2, common in freshwater fishes. Both are isoprenoid compounds containing a six-membered carbocyclic ring and an eleven-carbon side chain.

http://www.youtube.com/watch?v=dcw1m31zuTE

 

Vitamin A

Vitamin A consists of three biologically active molecules, retinol, retinal (retinaldehyde) and retinoic acid.

 

http://web.indstate.edu/thcme/mwking/alltransretinal.jpg

http://web.indstate.edu/thcme/mwking/cisretinal.jpg

All-trans-retinal

11-cis-retinal

 

http://web.indstate.edu/thcme/mwking/retinol.jpg

http://web.indstate.edu/thcme/mwking/retinoic.jpg

Retinol

Retinoic Acid

 

Carotenoids are provitamins of vitamin A. Carotenoids widely distributed in plants, particularly a-, b-, and g-carotene. The carotenes have no vitamin A activity but are converted into vitamin A by enzymatic reactions in the intestinal mucosa and the liver. b-Carotene, a symmetrical molecule, is cleaved in its center to yield two molecules of retinol. Retinol occurs in the tissues of mammals and is transported in the blood.

In vitamin A deficiency young persons fail to grow, the bones and nervous system fail to develop properly, the skin becomes dry and thickened, the kidneys and various glands degenerate, and both males and females become sterile.

mc1985(0923)

Although all tissues appear to be disturbed by vitamin A deficiency, the eyes are most conspicuously affected. In infants and young children the condition known as xerophthalmia ("dry eyes") is an early symptom of deficiency and is a common cause of blindness in some tropical areas where nutrition is generally poor. In adults an early sign of vitamin A deficiency is nightblindness, a deficiency in dark adaptation, which is often used as a diagnostic test.

Vitamin A Benefit

Detailed information is available on the role of vitamin A in the visual_cycle in vertebrates. The human retina contains two types of light-sensitive photoreceptor cells. Rod-cells are adapted to sensing low light intensities, but not colors; they are the cells involved in night vision, whose function is impaired by vitamin A deficiency. Cone cells, which sense colors, are adapted for high light intensities.

Retinal rod cells contain many membrane vesicles that serve as light receptors. About one-half of the protein in the membrane of these vesicles consists of the light-absorbing protein rhodopsin (visual purple). Rhodopsin consists of a protein, opsin, and tightly bound 11-cis-retinal, the aldehyde of vitamin A. When rhodopsin is exposed to light, the bound 11-cis-retinal undergoes transformation into all-trans-retinal, which causes a substantial change in the configuration of the retinal molecule. This reaction is nonenzymatic. The isomerization of retinal is followed by a series of other molecular changes, ending in the dissociation of the rhodopsin to yield free opsin and all-trans-retinal, which functions as a trigger setting off the nerve impulse.

11-cis-retinal all-trans-retinal

 

In order for rhodopsin to be regenerated from opsin and all-trans-retinal, the latter must undergo isomerization back to 11-cis-retinal. This appears to occur in a sequence of enzymatic reactions catalyzed by two enzymes:

 

retinal-reductase

all-trans-retinal + NADH + H+ all-trans-retinol + NAD+

retinol-isomerase

all-trans-retinol 11-cis-retinol

retinal-reductase

11-cis-retinol + NAD+ → 11-cis-retinal + NADH + H+

The 11-cis-retinal so formed now recombines with opsin to yield rhodopsin, thus completing the visual cycle.

Since vitamin A deficiency affects all tissues of mammals, not the retina alone, the role of retinal in the visual cycle does not represent the entire action of vitamin A. It appears possible that vitamin A may play a general role in:

- the transport of Ca2+ across certain membranes; such a more general role might explain the effects of vitamin A deficiency and excess on bony and connective tisues;

-         processes of growth and cell differentiation;

-         processes of glycoproteins formation whoch are the components of the biological mucosa .

The vitamin A requirement of man - 1,5-2 milligram per day.

Vitamin A is met in large part by green and yellow vegetables, such as lettuce, spinach, sweet potatoes, and carrots, which are rich in carotenes. Fish-liver oils are particularly rich in vitamin A. However, excessive intake of vitamin A is toxic and leads to easily fractured, fragile bones in children, as well as abnormal development of the fetus.

 

Vitamin D

Most important are vitamin D2, or ergocalciferol, and vitamin D3, or cholecalciferol, the form normally found in mammals. These compounds may be regarded as steroids.

It is now known that 7-dehydrocholesterol in the skin is the natural precursor of cholecalciferol in man; the conversion requires irradiation of the skin by sunlight. On a normal unsupplemented diet this is the major route by which people usually acquire vitamin D.

Vitamin D is a steroid hormone that functions to regulate specific gene expression following interaction with its intracellular receptor. The biologically active form of the hormone is 1,25-dihydroxy vitamin D3 (1,25-(OH)2D3, also termed calcitriol). Calcitriol functions primarily to regulate calcium and phosphorous homeostasis.

http://www.youtube.com/watch?v=JwPVibQ6_3Y&feature=related

http://www.youtube.com/watch?v=onSPZ0aBUKM&feature=related

http://www.youtube.com/watch?v=xwNhd2pQL0k&feature=related

 

http://web.indstate.edu/thcme/mwking/ergosterol.jpg

http://web.indstate.edu/thcme/mwking/vitamind2.jpg

Ergosterol

Vitamin D2

 

http://web.indstate.edu/thcme/mwking/7dehydrocholesterol.jpg

http://web.indstate.edu/thcme/mwking/vitamind3.jpg

7-Dehydrocholesterol

Vitamin D3

 

Cholecalciferol is converted into its derivative - 25-hydroxycholecalciferol. This product is more active biologically than cholecalciferol and it has been found to be the main circulating form of vitamin D in animals, formed in the liver. But 25-hydroxycholecalciferol was found to be metabolized further to 1,25-dihydroxycholecalciferol in kidneys. This compound is still more active; its administration produces rapid stimulation of Ca2+ absorption by the intestine.

http://web.indstate.edu/thcme/mwking/25hydroxyvitamind3.jpg

http://web.indstate.edu/thcme/mwking/1_25dihydroxyvitamind3.jpg

25-hydroxyvitamin D3

1,25-dihydroxyvitamin D3

 

So the kidney is the site of formation of 1,25-dihydroxycholecalciferol, which now appears to be the biologically active form of vitamin D, capable of acting directly on its major targets, the small intestine and the bones.

Vitamin D Deficit

1,25-dihydroxycholecalciferol promotes absorption of Ca2+ from the intestine into the blood, through its ability to stimulate the biosynthesis of specific protein(s) that participate in transport or binding of Ca2+ in the intestinal mucosa. This role of 1,25-dihydroxycholecalciferol is integrated with the action of parathyroid hormone. Whenever the Ca2+ concentration of the blood becomes lower than normal, the parathyroid glands secrete larger amounts of parathyroid hormone. This hormone acts on the kidney, stimulating it to produce more 1,25-dihydroxycholecalciferol from its precursor 25-hydroxycholecalciferol.

mc1984(0924)

Rickets, a disease of growing bone, is developed in the deficiency of vitamin D in organism.

http://www.youtube.com/watch?v=n7vybcT9_F4

 

As with vitamin A, excessive intake of vitamin D causes the bones to become fragile and to undergo multiple fractures, suggesting that both vitamins play a role in biological transport and deposition of calcium.

Vitamin D Benefit

Most natural foods contain little of vitamin D; vitamin D in the diet comes largely from fish-liver oils, liver, yoke of eggs, butter. Vitamin D preparations available commercially are products of the ultraviolet irradiation of ergosterol from yeast.

About 2,5-10 mkg of vitamin D is required by an adult daily and 12-25 mkg by children. The vitamin can be stored in sufficient amounts in the liver for a single dose to suffice for some weeks.

Vitamin E

http://web.indstate.edu/thcme/mwking/alphatocopherol.jpg

a-Tocopherol

 

Vitamin E was first recognized as a factor in vegetable oils that restores fertility in rats grown on cow's milk alone and otherwise incapable of bearing young. It was isolated from wheat germ and was given the name tocopherol. Several different tocopherols having vitamin E activity have been found in plants; the most active and abundant is a-tocopherol.

Vitamin E Benefit

The deficiency of tocopherol produces many other symptoms besides infertility in male and female, e.g., degeneration of the kidneys, the deposition of brown pigments in lipid depots, necrosis of the liver, and dystrophy, or wasting, of skeletal muscles.

Vitamin E and Heart Disease

Tocopherols have been found to have antioxidant activity; i.e., they prevent the autoxidation of highly unsaturated fatty acids when they are exposed to molecular oxygen. One of the functions of tocopherol may be to protect highly unsaturated fatty acids in the lipids of biological membranes against the deleterious effects of molecular oxygen. Normally, autoxidation products of unsaturated fats do not occur in the tissues, but in tocopherol deficiency they are detectable in the fat depots, liver, and other organs.

Due to the hydrophobic side radical tocopherol can be built into the phospholipid matrix of biological membranes and stabilize the mobility and microviscosity of membrane proteins and lipids.

Tocopherol is the most potent natural antioxidant.

About 10-20 mg of vitamin E is required per day.

The most abundant sources of vitamin E are oils (sunflower, corn, soybean oils), fresh vegetables, animal stuffs (meat, butter, egg yoke).

http://www.youtube.com/watch?v=8oRUF_g-J3k&feature=related

 

Vitamin K

The K vitamins exist naturally as K1 (phylloquinone) in green vegetables and K2 (menaquinone) produced by intestinal bacteria and K3 is synthetic menadione. When administered, vitamin K3 is alkylated to one of the vitamin K2 forms of menaquinone.

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Vitamin K1

Vitamin K2


Vitamin

 

Vitamin K was first discovered as a nutritional factor required for normal blood-clotting time. At least two forms of vitamin K are known; vitamin K2 is believed to be the active form. Vitamin K deficiency cannot readily be produced in rats and other mammals because the vitamin is synthesized by intestinal bacteria.

http://www.youtube.com/watch?v=DVGsnlVCoeA

http://www.youtube.com/watch?v=WI24c2LYFug&feature=related

 

The only known result of vitamin K deficiency is a failure in the biosynthesis of the enzyme proconvertin in the liver. This enzyme catalyzes a step in a complex sequence of reactions involved in the formation of prothrombin, the precursor of thrombin, a protein that accelerates the conversion of fibrinogen into fibrin, the insoluble protein constituting the fibrous portion of blood clots.

Vitamin K Benefit

The compound dicumarol, an analog of vitamin K, produces symptoms in animals resembling vitamin K deficiency; it is believed to block the action of vitamin K. Dicumarol is used in clinical medicine to prevent clotting in blood vessels. Dicumarol is the antivitamin of vitamin K.

Some evidence indicates that vitamin K may function as a coenzyme in a specialized route of electron transport in animal tissues; since vitamin K is a quinone which can be reduced reversibly to a quinol, it may serve as an electron carrier.

Function

  • aids in reducing excessive menstrual flow
  • aids the absorption of calcium in bone
  • essential for normal liver functioning
  • essential for synthesis of four proteins that act in coagulation
  • important in maintaining vitality and longevity
  • necessary for formation of prothrombin which is required for  effective blood clotting
  • involved in electron transport mechanism and oxdative phosphorylation

Food Source

  • alfalfa
  • blackstrap molasses
  • broccoli
  • Brussels sprouts
  • cauliflower
  • cereals
  • cow's milk
  • egg yolks
  • fish liver oils
  • green plants, such as lettuce
  • kelp
  • leafy green vegetables, such as cabbage, spinach
  • meats, such as pig and beef liver
  • peas
  • polyunsaturated oils
  • potatoes
  • string beans
  • yogurt

Effective With

Increased Intakes Needed

  • after prolonged paraffin ingestion
  • for those with biliary obstructions
  • for those with liver disease
  • if taking antibiotics for long duration
  • if you have a malabsorption disease
  • in newborn babies
  • in overdose of anticoagulant drugs, such as Warfarin, Dicoumarol, which neutralize the effect of Vitamin K

Used For

  • anticoagulant drug overdose
  • hemorrhagic disease in newborn babies
  • inhibiting some cancer tumors
  • overcoming inability to absorb vitamins
  • overcoming effects of antibiotics on intestinal bacteria
  • protection against osteoporosis

Destroyed By

  • acids
  • alkalis
  • commercial processing
  • light and ultra-violet irradiation
  • oxidizing agents

Symptoms of Deficiency

  • excessive bleeding and hemorrhage

In babies:

  • bleeding from the stomach, intestines, umbilical cord site

Deficiency Caused By

In Babies:

  • low levels in human breast milk
  • poor transfer across placenta
  • sterile intestine with no bacteria

In Adults:

  • as a consequence of sprue
  • Celiac's Disease
  • destruction of intestinal bacteria by antibiotics
  • lack of bile salts
  • liver conditions, such as viral hepatitis
  • surgical removal of intestines
  • prolonged ingestion of liquid paraffin

Deficiency Leads To

  • inability of blood to coagulate

 

Hypovitaminos of vitamin K in man can be developed in liver diseases when there is the decrease of bile acids amount in intestine and as result the inhibition of fat soluble substances absorption is observed.

Vitamin K is produced by many microorganisms in the intestine. also Plants (cabbage, tomato, lettuce)are natural sources of vitamin K.

Adult person requires 200-300 mkg of vitamin K per day.

References:

1.    John Mc Murry, Mary E. Castellion. General, Organic and Biological Chemistry.- New Jersy: Prentice Hall, 1992.- 764 p.

2.    John W. Suttie. Introduction to Biochemistry. New York: Holt, Rinehart and Winston, Inc., 1992.- 364 p.

3.    Robert K. Murray, Daryl K. Granner. Harpers illustrated Biochemistry. India: International Education, 2003.- 693 p.

4.    VK Malhotra. Biochemistry for students. India: Jaypee Brothers, Medical Publishers LTD, 1998. 334p.

5.    Lehninger A. Principles of Biochemistry. New York: Worth Publishers, Inc., 1982. 1010 p.

6.    Stryer L. Biochemistry. New York: W.H.Freeman and Company, 1988. 1086 p

 

Investigation of water soluble (coenzyme) vitamins functional role in metabolism and cell functions realization.

Vitamins are nutrients required in tiny amounts for essential metabolic reactions in the body. The term vitamin does not include other essential nutrients such as dietary minerals, essential fatty acids, or essential amino acids, nor does it encompass the large number of other nutrients that promote health but that are not essential for life.

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Vitamins are bio-molecules that act both as catalysts and substrates in chemical reactions. When acting as a catalyst, vitamins are bound to enzymes and are called cofactors. (For example, vitamin K forms part of the proteases involved in blood clotting.) Vitamins also act as coenzymes to carry chemical groups between enzymes. (For example, folic acid carries various forms of carbon groupsmethyl, formyl or methylenein the cell.)/

vitaminsUntil the 1900s, vitamins were obtained solely through food intake. Many food sources contain different ratios of vitamins. Therefore, if the only source of vitamins is food, changes in diet will alter the types and amounts of vitamins ingested. However, as many vitamins can be stored by the body, short-term deficiencies (which, for example, could occur during a particular growing season) do not usually cause disease.

Vitamins have been produced as commodity chemicals and made widely available as inexpensive pills for several decades,[2] allowing supplementation of the dietary intake.

 

Difference from water soluble vitamins: water soluble vitamins are included into coenzymes, don't have provitamins, are not included into the membranes, and hypervitaminoses are not peculiar for them.

With exception of vitamin B6 and B12, they are readily excreted in urine without appreciable storage, so frequent consumption becomes necessary. They are generally nontoxic when present in excess of needs, although symptoms may be reported in people taking megadoses of niacin, vitamin C, or pyridoxine (vitamin B6). All the B vitamins function as coenzymes or cofactors, assisting in the activity of important enzymes and allowing energy-producing reactions to proceed normally. As a result, any lack of water-soluble vitamins mostly affects growing or rapidly metabolizing tissues such as skin, blood, the digestive tract, and the nervous system. Water-soluble vitamins are easily lost with overcooking.

 

Water-soluble vitamins and their characteristics.

 

 

Common food sources

Major functions

Deficiency symptoms

Overconsumption symptoms

Stability in foods

 

 

Vitamin C (abscorbic acid)

 

 

Citrus fruits, broccoli, strawberries, melon, green pepper, tomatoes, dark green vegetables, potatoes.

Formation of collagen (a component of tissues), helps hold them together; wound healing; maintaining blood vessels, bones, teeth; absorption of iron, calcium, folacin; production of brain hormones, immune factors; antioxidant.

Bleeding gums; wounds don't heal; bruise easily; dry, rough skin; scurvy; sore joints and bones; increased infections.

Nontoxic under normal conditions; rebound scurvy when high doses discontinued; diarrhea, bloating, cramps; increased incidence of kidney stones.

Most unstable under heat, drying, storage; very soluble in water, leaches out of some vegetables during cooking; alkalinity (baking soda) destroys vitamin C.

 

 

Thiamin (vitamin B1 )

 

 

Pork, liver, whole grains, enriched grain products, peas, meat, legumes.

Helps release energy from foods; promotes normal appetite; important in function of nervous system.

Mental confusion; muscle weakness, wasting; edema; impaired growth; beriberi.

None known.

Losses depend on cooking method, length, alkalinity of cooking medium; destroyed by sulfite used to treat dried fruits such as apricots; dissolves in cooking water.

 

 

Riboflavin (vitamin B2)

 

 

Liver, milk, dark green vegetables, whole and enriched grain products, eggs.

Helps release energy from foods; promotes good vision, healthy skin.

Cracks at corners of mouth; dermatitis around nose and lips; eyes sensitive to light.

None known.

Sensitive to light; unstable in alkaline solutions.

 

Niacin (nicotinamide, nicotinic acid)

 

 

Liver, fish, poultry, meat, peanuts, whole and enriched grain products.

Energy production from foods; aids digestion, promotes normal appetite; promotes healthy skin, nerves.

Skin disorders; diarrhea; weakness; mental confusion; irritability.

Abnormal liver function; cramps; nausea; irritability.

 

 

Vitamin B6 (pyridoxine, pyridoxal, pyridoxamine)

 

 

Pork, meats, whole grains and cereals, legumes, green, leafy vegetables.

Aids in protein metabolism, absorption; aids in red blood cell formation; helps body use fats.

Skin disorders, dermatitis, cracks at corners of mouth; irritability; anemia; kidney stones; nausea; smooth tongue.

None known.

Considerable losses during cooking.

 

 

 

 

 

 

 

 

 

 


 

Folacin (folic acid)

 

Liver, kidney, dark green leafy vegetables, meats, fish, whole grains, fortified grains and cereals, legumes, citrus fruits.

Aids in protein metabolism; promotes red blood cell formation; prevents birth defects of spine, brain; lowers homocystein levels and thus coronary heart disease risk.

Anemia; smooth tongue; diarrhea.

May mask vitamin B12 deficiency (pernicious anemia).

Easily destroyed by storing, cooking and other processing.

 

Vitamin B12

 

Found only in animal foods: meats, liver, kidney, fish, eggs, milk and milk products, oysters, shellfish.

Aids in building of genetic material; aids in development of normal red blood cells; maintenance of nervous system.

Pernicious anemia, anemia; neurological disorders; degeneration of peripheral nerves that may cause numbness, tingling in fingers and toes.

None known.

 

 

Pantothenic acid

 

Liver, kidney, meats, egg yolk, whole grains, legumes; also made by intestinal bacteria.

Involved in energy production; aids in formation of hormones.

Uncommon due to availability in most foods; fatigue; nausea, abdominal cramps; difficulty sleeping.

None known.

About half of pantothenic acid is lost in the milling of grains and heavily refined foods.

 

 

Thiamin (Vitamin B1)

103

Thiamine or thiamin, also known as vitamin B1, is a colorless compound with chemical formula C12H17N4OS. It is soluble in water and insoluble in alcohol.

Thiamine decomposes if heated. Its chemical structure contains a pyrimidine ring and a thiazole ring.Thiamine was first discovered in 1910 by Umetaro Suzuki in Japan when researching how rice bran cured patients of Beriberi. He named it aberic acid. Thiamine diphosphate (ThDP) or thiamine pyrophosphate (TPP) is a coenzyme for pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched-chain

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Thiamin pyrophosphate

alpha-keto acid dehydrogenase, and transketolase. The first two of these enzymes function in the metabolism of carbohydrates, while transketolase functions in the pentose phosphate pathway to synthesize NADPH and the pentose sugars deoxyribose and ribose. In general, TPP functions as a cofactor for enzymes that catalyze the dehydrogenation (decarboxylation and subsequent conjugation to Coenzyme A) of alpha-keto acids. TPP is synthesized by the enzyme thiamine

pyrophosphokinase, which requires free thiamine, magnesium, and adenosine triphosphate.

Good sources: Thiamine is found naturally in the following foods, each of which contains at least 0.1mg of the vitamin per 28-100g (1-3.5oz): Green peas, Spinach, Liver, Beef, Pork, Navy beans, Nuts, Pinto beans, Soybeans, Whole-grain and Enriched Cereals, Breads, Yeast, and Legumes. http://rds.yahoo.com/_ylt=A9gnMikzjRJGREcA5nCjzbkF;_ylu=X3oDMTA4NDgyNWN0BHNlYwNwcm9m/SIG=12nbk4k5o/EXP=1175707315/**http%3A/www.pharmaton.ch/Images/illustrations/2_2_1_2_vitamin_b1.jpghttp://rds.yahoo.com/_ylt=A9gnMilojBJGDkkAUSSjzbkF;_ylu=X3oDMTA4NDgyNWN0BHNlYwNwcm9m/SIG=12i7hcuhv/EXP=1175707112/**http%3A/www.doctorsecrets.com/your-diet/vitamin-food-source.gif

http://www.youtube.com/watch?v=Q5BCmsixuqM

 

Thiamin functions as the coenzyme thiamin pyrophosphate (TPP) in the metabolism of carbohydrate and in conduction of nerve impulses. Thiamin deficiency causes beri-beri, which is frequently seen in parts of the world where polished (white) rice or unenriched white flour are predominantly eaten.

105

http://www.youtube.com/watch?v=PD_CoEngu4M&feature=related

 

There are three basic expressions of beriberi: childhood, wet, and dry. Childhood beriberi stunts growth in infants and children. Wet beriberi is the classic form, with swelling due to fluid retention (edema) in the lower limbs that spreads to the upper body, affecting the heart and leading to heart failure. Dry beriberi affects peripheral nerves, initially causing tingling or burning sensations in the lower limbs and progressing to nerve degeneration,

Vitamin B1 Benefit

muscle wasting, and weight loss. Thiamine-deficiency disease in North America commonly occurs in people with heavy alcohol consumption and is called Wernicke-Korsakoff syndrome. It is caused by poor food intake and by decreased absorption and increased excretion caused by alcohol consumption.

 

Riboflavin (Vitamin B2)

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Riboflavin is stable when heated in ordinary cooking, unless the food is exposed to ultraviolet radiation (sunlight). To prevent riboflavin breakdown, riboflavin-rich foods such as milk, milk products, and cereals are packaged in opaque containers. Riboflavin is a component of two coenzymesflavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD)that act as hydrogen carriers when carbohydrates and fats are used to produce energy. It is helpful in maintaining good vision and healthy hair, skin and nails, and it is necessary for normal cell growth.

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Thiamin pyrophosphate

Riboflavin deficiency causes a condition known as ariboflavinosis, which is marked by cheilosis (cracks at the corners of the mouth), oily scaling of the skin, and a red, sore tongue. In addition, cataracts may occur more frequently with riboflavin deficiency. A deficiency of this nutrient is usually a part of multinutrient deficiency and does not occur in isolation. In North America, it is mostly observed in alcoholics, elderly persons with low income or depression, and people with poor eating habits, particularly those who consume highly refined and fast foods and those who do not consume milk and milk products.

http://rds.yahoo.com/_ylt=A9gnMiHyjRJGyQoBiFujzbkF;_ylu=X3oDMTA4NDgyNWN0BHNlYwNwcm9m/SIG=12e8hkifv/EXP=1175707506/**http%3A/www.apotheke-am-buchenberg.de/bilder/vitamin-b2.jpg

http://www.youtube.com/watch?v=qpvNaGIJMzw

Unlike fat-soluble vitamins, water-soluble vitamins are easily lost during cooking and processing. The body does not store excess quantities of most water-soluble vitamins, so foods bearing them must be consumed frequently.

Niacin (Vitamin B5)

Niacin

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Nicotinamide

Nicotinic Acid

 

Niacin exists in two forms, nicotinic acid and nicotinamide. Both forms are readily absorbed from the stomach and the small intestine. Niacin is stored in small amounts in the liver and transported to tissues, where it is converted to coenzyme forms. Any excess is excreted in urine. Niacin is one of the most stable of the B vitamins. It is resistant to heat and light, and to both acid and alkali environments. The human body is capable of converting the amino acid tryptophan to niacin when needed. However, when both tryptophan and niacin are deficient, tryptophan is used for protein synthesis.

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Structure of NAD+

There are two coenzyme forms of niacin: nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phophate (NADP+). They both help break down and utilize proteins, fats, and carbohydrates for energy. Niacin is essential for growth and is involved in hormone synthesis.

Pellagra results from a combined deficiency of niacin and tryptophan. Long-term deficiency leads to central nervous system dysfunction manifested as confusion, apathy, disorientation, and eventually coma and death. Pellagra is rarely seen in industrialized countries, where it may be observed in people with rare disorder of tryptophan metabolism (Hartnup's disease), alcoholics, and those with diseases that affect food intake.

http://www.youtube.com/watch?v=UrDeVyiXzyg&feature=related

The liver can synthesize niacin from the essential aminoacid tryptophan, but the synthesis is extremely slow; 60 mg of tryptophan are required to make one milligram of niacin. Dietary niacin deficiency tends to occur only in areas where people eat corn, the only grain low in niacin, as a staple food, and that don't use lime during maize (corn) meal/flour production. Alkali lime releases the tryptophan from the corn so that it can be absorbed in the gut, and converted to niacin.

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Niacin plays an important role in the production of several sex and stress-related hormones, particularly those made by the adrenal gland. Niacin, when taken in large doses, increases the level of high density lipoprotein (HDL) or "good" cholesterol in blood, and is sometimes prescribed for patients with low HDL, and at high risk of heart attack. Niacin (but not niacinamide) is also used in the treatment of hyperlipidemia because it reduces very low density lipoprotein (VLDL), a precursor of low density lipoprotein (LDL) or "bad" cholesterol, secretion from the liver, and inhibits cholesterol synthesis. Vitamin B3 Benefit

The main problem with the clinical use of niacin for dyslipidemia is the occurrence of skin flushing, even with moderate doses.

Recommended intake is expressed as milligrams of niacin equivalents (NE) to account for niacin synthesized from tryptophan. High doses taken orally as nicotinic acid at 1.5 to 2 grams per day can decrease cholesterol and triglyceride levels, and along with diet and exercise can slow or reverse the progression of heart disease. " No Flush vitamin b3, niacin.(Strenght  not  exactly  as  Shown  on  bottle.) "

The nicotinamide form of niacin in multivitamin and B-complex tablets do not work for this purpose. Supplementation should be under a physician's guidance.

http://www.youtube.com/watch?v=MLFZ8CsrJqU&feature=related

 

Pantothenic Acid (Vitamin B3)

Pantothenic Acid

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Pantothenic Acid

 

Pantothenic acid, also called vitamin B3, is a water-soluble vitamin required to sustain life. Pantothenic acid is needed to form coenzyme-A (CoA), and is critical in the metabolism and synthesis of carbohydrates, proteins, and fats. Its name

is derived from the Greek pantothen meaning "from everywhere" and small quantities of pantothenic acid are found in nearly every food, with high amounts in

whole grain cereals, legumes, eggs, meat, and royal jelly

Pantothenic acid is stable in moist heat. It is destroyed by vinegar (acid), baking soda (alkali), and dry heat. Significant losses occur during the processing and refining of foods. Pantothenic acid is released from coenzyme A in food in the small intestine. After absorption, it is transported to tissues, where coenzyme A is resynthesized. Coenzyme A is essential for the formation of energy as adenosine triphosphate (ATP) from carbohydrate, protein, alcohol, and fat.

 

Coenzyme A is also important in the synthesis of fatty acids, cholesterol, steroids, and the neurotransmitter acetylcholine, which is essential for transmission of nerve impulses to muscles.

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Coenzyme A

 

Dietary deficiency occurs in conjunction with other B-vitamin deficiencies. Pantothenic acid is used in the synthesis of coenzyme A (abbreviated as CoA). Coenzyme A may act as an acyl group carrier to form acetyl-CoA and other related compounds; this is a way to transport carbon atoms within the cell. The transfer of carbon atoms by coenzyme A is important in cellular respiration, as well as the biosynthesis of many important compounds such as fatty acids, cholesterol, and acetylcholine. Dietary deficiency occurs in conjunction with other B-vitamin deficiencies. In studies, experimentally induced deficiency in humans has resulted in headache, fatigue, impaired muscle coordination, abdominal cramps, and vomiting.

In studies, experimentally induced deficiency in humans has resulted in headache, fatigue, impaired muscle coordination, abdominal cramps, and vomiting.

Biotin (Vitamin B8)

Biotin

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Biotin is a water soluble vitamin and a member of Vitamin B complex.  Also known as Vitamin H, Bios II, Co-enzyme R.  Its natural form is D-biotin.  It was isolated from liver in 1941 by Dr. Paul Gyorgy.

http://www.youtube.com/watch?v=o9lJoKoF4DE

FUNCTION

  • co-enzyme in wide variety of body metabolic reactions
  • needed for production of energy from carbohydrates, fats and proteins
  • needed for interconversions
  • essential for maintenance of healthy skin, hair, sweat glands, nerves, bone marrow and glands producing sex hormones

FOOD SOURCE

  • Brewer's Yeast
  • cheese
  • eggs
  • maize
  • fish, fatty, white
  • meats, especially pig liver and kidney
  • milk
  • oats
  • wheat bran
  • wheat germ
  • wholemeal grains
  • unpolished brown rice
  • vegetables
  • yoghurt

EFFECTIVE WITH

INCREASED INTAKES NEEDED

  • by newborn children being fed on dried milk
  • during stress situations
  • when on antibiotic therapy

USED FOR

  • seborrheic dermatitis
  • Leiner's Disease
  • alopecia (hair falling out in handfuls)
  • scalp disease
  • skin complaints
  • preventing cot death (given to babies)

DESTROYED BY

  • leaching into cooking
  • drying of milk for baby foods

SYMPTOMS OF DEFICIENCY

In babies:

  • dry scaling of the scalp and face
  • persistent diarrhea

In adults:

  • depression
  • diminished reflexes
  • fatigue
  • hair loss
  • increase in blood cholesterol levels
  • loss of appetite
  • muscular pains
  • nausea
  • pale, smooth tongue
  • sleepiness

DEFICIENCY LEADS TO

  • specific anemia
  • deficiency may be induced by excessive intake of raw egg whites, which contain the protein Avidin which immobilizes Biotin

SYMPTOMS OF TOXICITY

  • toxicity unknown

High quality Vitamin B (Biotin) can be purchased from Global Herbal Supplies

Biotin

 

Biotin is the most stable of B vitamins. It is commonly found in two forms: the free vitamin and the protein-bound coenzyme form called biocytin. Biotin is absorbed in the small intestine, and it requires digestion by enzyme biotinidase, which is present in the small intestine. Biotin is synthesized by bacteria in the large intestine, but its absorption is questionable. Biotincontaining coenzymes participate in key reactions that produce energy from carbohydrate and synthesize fatty acids and protein.

Avidin is a protein in raw egg white, which can bind to the biotin in the stomach and decrease its absorption. Therefore, consumption of raw whites is of concern due to the risk of becoming biotin deficient. Cooking the egg white, however, destroys avidin. Deficiency may develop in infants born with a genetic defect that results in reduced levels of biotinidase. In the past, biotin deficiency was observed in infants fed biotin-deficient formula, so it is now added to infant formulas and other baby foods.

Vitamin B6

Vitamin B6

 

http://web.indstate.edu/thcme/mwking/pyridoxal.jpg

 

 

Pyridoxal

 

http://www.youtube.com/watch?v=zwUc5gHoF_U&feature=related

 

Pyridoxal, pyridoxamine and pyridoxine are collectively known as vitamin B6. All three compounds are efficiently converted to the biologically active form of vitamin B6, pyridoxal phosphate. This conversion is catalyzed by the ATP requiring enzyme, pyridoxal kinase.

http://web.indstate.edu/thcme/mwking/pyridoxalphosphate.jpg

Pyridoxal Phosphate

http://www.youtube.com/watch?v=Q9yBs6wvMFc

 

Vitamin B6 is present in three forms: pyridoxal, pyridoxine, and pyridoxamine. All forms can be converted to the active vitamin-B6 coenzyme in the body. Pyridoxal phosphate (PLP) is the predominant biologically active form. Vitamin B6 is not stable in heat or in alkaline conditions, so cooking and food processing reduce its content in food. Both coenzyme and free forms are absorbed in the small intestine and transported to the liver, where they are phosphorylated and released into circulation, bound to albumin for transport to tissues. Vitamin B6 is stored in the muscle and only excreted in urine when intake is excessive.

Vitamin B6 Benefit

PLP participates in amino acid synthesis and the interconversion of some amino acids. It catalyzes a step in the synthesis of hemoglobin, which is needed to transport oxygen in blood. PLP helps maintain blood glucose levels by facilitating the release of glucose from liver and muscle glycogen. It also plays a role in the synthesis of many neurotransmitters important for brain function. This has led some physicians to prescribe megadoses of B6 to patients with psychological problems such as depression and mood swings, and to some women for premenstrual syndrome (PMS). It is unclear, however, whether this therapy is effective. PLP participates in the conversion of the amino acid tryptophan to niacin and helps avoid niacin deficiency. Pyridoxine affects immune function, as it is essential for the formation of a type of white blood cell.

Populations at risk of vitamin-B6 deficiency include alcoholics and elderly persons who consume an inadequate diet. Individuals taking medication to treat Parkinson's disease or tuberculosis may take extra vitamin B6 with physician supervision. Carpal tunnel syndrome, a nerve disorder of the wrist, has also been treated with large daily doses of B6. However, data on its effectiveness are conflicting.

Folic Acid, Folate, Folacin (Vitamin B9)

Image:Folic-acid-3D-vdW.pngFolic Acid

http://web.indstate.edu/thcme/mwking/folate.jpg

 

Folacin or folate, as it is usually called, is the form of vitamin B9 naturally present in foods, whereas folic acid is the synthetic form added to fortified foods and supplements. Both forms are absorbed in the small intestine and stored in the liver. The folic acid form, however, is more efficiently absorbed and available to the body. When consumed in excess of needs, both forms are excreted in urine and easily destroyed by heat, oxidation, and light.

Vitamin B9 Benefits

Folic acid is a water soluble vitamin and is a member of the Vitamin B complex. Also known as Folacin, pteroyl-L-glutamic acid (PGA), vitamin Bc or vitamin M. Folic acid and its derivatives (mostly the tri and heptaglutamyl peptides) are widespread in nature. It is a specific growth factor for certain micro-organisms.  Found in yeast and liver in 1935.

All forms of this vitamin are readily converted to the coenzyme form called tetrahydrofolate (THFA), which plays a key role in transferring single-carbon methyl units during the synthesis of DNA and RNA, and in interconversions of amino acids. Folate also plays an important role in the synthesis of neurotransmitters. Meeting folate needs can improve mood and mental functions.

Function

  • involved in the formation of new cells
  • involved in the metabolism of ribonucleic acids (RNA) and deoxyribonucleic acids (DNA), essential for protein synthesis, formation of blood and transmission of genetic code
  • essential during pregnancy to reduce the risk of neural tube defects (birth defects affecting the brain and/or spinal cord)essential for the normal growth and development of the fetus
  • involved in the biosynthesis of purines, serines and glycine
  • involved in some functions associated with Vitamin B12
  • necessary for building resistance to diseases in the thymus gland of new born babies and infants
  • may reduce the risk of cervical dysplasia
  • necessary for red blood cell production

Food Source

  • bananas
  • Brewers's Yeast
  • citrus fruits, peeled
  • eggs
  • fatty fish
  • fresh nuts
  • green leafy vegetables
  • meats, especially pig liver and kidney
  • milk
  • oats
  • pulses, such as lentils
  • roasted nuts
  • soy products, such as tofu
  • unpolished brown rice
  • wheat germ
  • wheat bran
  • wheat grains

Effective With

  • B-Complex
  • B12
  • Biotin
  • Pantothenic Acid
  • Vitamin C

Increased Intakes Needed

  • by alcohol drinkers
  • by the elderly
  • during pregnancy and breastfeeding
  • if taking contraceptive pill
  • if taking the drugs, Aspirin, Cholestyramine,  Isethionate, Isoniazid, Methotrexate,  Pentamidime, Phenytoin (may be neutralized), Primidone, Pyrimethamine, Triamterene,Trimethoprim

Used For

  • malabsorption in geriatric patients
  • megaloblastic anemia
  • mental deterioration
  • psychosis
  • schizophrenia

Destroyed By

  • leached into cooking water
  • processing and cooking of vegetables, fruits and dairy products
  • unstable to oxygen at high temperatures but protected by Vitamin C

Symptoms of Deficiency

  • breathlessness
  • fatigue
  • irritability
  • sleeplessness
  • weakness

Deficiency Leads To

Various conditions relating to childbirth:

  • abortion
  • birth defects, such as neural tube defect which causes spina bifida
  • hemorrhage following birth
  • premature birth
  • premature separation of the placenta from the uterus
  • toxemia

As well as:

  • megaloblastic anemia (red blood cells are large and uneven with a shortened life span)
  • mild mental symptoms, such as forgetfulness and confusion

Symptoms of Toxicity

Folic Acid has a low toxicity but occasionally the following symptoms occur:

  • abdominal distension
  • flatulence (gas/wind)
  • irritability
  • loss of appetite
  • nausea
  • over-activity
  • sleep disturbance
  • symptoms of fever
  • temperature rise

Long term high doses may cause Vitamin B12 losses from the body

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Folate deficiency is one of the most common vitamin deficiencies. Early symptoms are nonspecific and include tiredness, irritability, and loss of appetite. Severe folate deficiency leads to macrocytic anemia, a condition in which cells in the bone marrow cannot divide normally and red blood cells remain in a large immature form called macrocytes. Large immature cells also appear along the length of the gastrointestinal tract, resulting in abdominal pain and diarrhea.

Vitamin B9 Source

Pregnancy is a time of rapid cell multiplication and DNA synthesis, which increases the need for folate. Folate deficiency may lead to neural tube defects such as spina bifida (failure of the spine to close properly during the first month of pregnancy) and anencephaly (closure of the neural tube during fetal development, resulting in part of the cranium not being formed). Seventy percent of these defects could be avoided by adequate folate status before conception, and it is recommended that all women of childbearing age consume at least 400 micrograms (μg) of folic acid each day from fortified foods and supplements. Other groups at risk of deficiency include elderly persons and persons suffering from alcohol abuse or taking certain prescription drugs.

Vitamin B12

Vitamin B12 is found in its free-vitamin form, called cyanocobalamin, and in two active coenzyme forms. Absorption of vitamin B12 requires the presence of intrinsic factor,

Vitamin B12 Benefits

a protein synthesized by acid-producing cells of the stomach. The vitamin is absorbed in the terminal portion of the small intestine called the ileum. Most of body's supply of vitamin B12 is stored in the liver.

Vitamin B12

 

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Cyanocobalamin

 

Vitamin B12 is defficiently conserved in the body, since most of it is secreted into bile and reabsorbed. This explains the slow development (about two years) of deficiency in people with reduced intake or absorption. Vitamin B12 is stable when heated and slowly loses its activity when exposed to light, oxygen, and acid or alkaline environments.

Vitamin B12 Eczema

 

Vitamin B12 coenzymes help recycle folate coenzymes involved in the synthesis of DNA and RNA, and in the normal formation of red blood cells. Vitamin B12 prevents degeneration of the myelin sheaths that cover nerves and help maintain normal electrical conductivity through the nerves.

http://web.indstate.edu/thcme/mwking/folate-derivatives.jpg

Active center of tetrahydrofolate (THF). Note that the N5 position is the site of attachment of methyl groups, the N10 the site for attachment of formyl and formimino groups and that both N5 and N10 bridge the methylene and methenyl groups

 

Vitamin-B12 deficiency results in pernicious anemia, which is caused by a genetic problem in the production of intrinsic factor. When this occurs, folate function is impaired, leading to macrocytic anemia due to interference in normal DNA synthesis. Unlike folate deficiency, the anemia caused by vitamin-B12 deficiency is accompanied by symptoms of nerve degeneration, which if left untreated can result in paralysis and death.

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Since vitamin B12 is well conserved in the body, it is difficult to become deficient from dietary factors alone, unless a person is a strict vegan and consumes a

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diet devoid of eggs and dairy for several years. Deficiency is usually observed when B12 absorption is hampered by disease or surgery to the stomach or ileum, damage to gastric mucosa by alcoholism, or prolonged use of anti-ulcer medications that affect secretion of intrinsic factor. Agerelated decrease in stomach-acid production also reduces absorption of B12 in elderly persons. These groups are advised to consume fortified foods or take a supplemental form of vitamin B12.

Choline

Choline-skeletal

For many years, choline was not considered a vitamin because the body makes enough of it to meet its needs in most age groups. However, research now shows that choline production in the body is not enough to cover requirements. Choline is not considered a B vitamin because it does not have a coenzyme function and the amount in the body is much greater than other B vitamins. Choline not only helps maintain the structural integrity of membranes surrounding every cell in the body, but also can play a role in nerve signaling, cholesterol transport, and energy metabolism. An "adequate intake" is 550 milligrams per day for men and 425 milligrams per day for women. Choline is widely found in foods, so it is unlikely that a dietary deficiency will occur.

 

Vitamin C (Ascorbic Acid)

Ascorbic Acid

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In 1746, James Lind, a British physician, conducted the first nutrition experiment on human beings in an effort to find a cure for scurvy.

James Lind

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James Lind (1716  1794),a British Royal Navy surgeon who, in 1774, identified that a quality in fruit prevented the disease of scurvy in what was the first recorded controlled experiment

However, it was not until nearly 200 years later that ascorbic acid, or vitamin C, was discovered. Vitamin C participates in many reactions by donating electrons as hydrogen atoms. In a reducing reaction, the electron in the hydrogen atom donated by vitamin C combines with other participating molecules, making vitamin C a reducing agent, essential to the activity of many enzymes. By neutralizing free radicals, vitamin C may reduce the risk of heart disease, certain forms of cancer, and cataracts.

Vitamin C is needed to form and maintain collagen, a fibrous protein that gives strength to connective tissues in skin, cartilage, bones, teeth, and joints. Collagen is also needed for the healing of wounds.

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When added to meals, vitamin C increases intestinal absorption of iron from plant-based foods. High concentration of vitamin C in white blood cells enables the immune system to function properly by providing protection against oxidative damage from free radicals generated during their action against bacterial, viral, or fungal infections.

Vitamin C Deficit

Vitamin C also recycles oxidized vitamin E for reuse in cells, and it helps folic acid convert to its active form, (THF). Vitamin C helps synthesize carnitine, adrenaline, epinephrine, the neurotransmitter serotonin, the thyroid hormone thyroxine, bile acids, and steroid hormones.

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A deficiency of vitamin C causes widespread connective tissue changes throughout the body. Deficiencies may occur in people who eat few fruits and vegetables, follow restrictive diets, or abuse alcohol and drugs. Smokers also have lower vitamin-C status. Supplementation may be prescribed by physicians to speed the healing of bedsores, skin ulcers, fractures, burns, and after surgery. Research has shown that doses up to 1 gram per day may have small effects on duration and severity of the common cold, but not on the prevention of its occurrence.

Ascorbic acid

Ascorbic acid is required in the diet of only a few vertebrates man, monkeys, the guinea pig, and certain fishes. Some insects and other invertebrates also require ascorbic acid, but most other higher animals and plants can synthesize ascorbic acid from glucose or other simple precursors. Ascorbic acid is not present in microorganisms, nor does it seem to be required.

Ascorbic acid is a strong reducing agent, readily losing hydrogen atoms to become dehydroascorbic acid, which also has vitamin C activity. However, vitamin activity is lost when the lactone ring of dehydroascorbic acid is hydrolyzed to yield diketogulonic acid.

Ascorbic Dehydroascorbic Diketogulonic

acid acid acid

Biological role of ascorbic acid:

-         acts as a cofactor in the enzymatic hydroxylation of proline to hydroxyproline and in other hydroxylation reactions;

-         inhibits the oxidation of hemoglobin;

-         accelerates the oxidation of glucose in pentose phosphate pathway;

-         reduces the disulfide bonds to sulfhydryl bonds;

-         is necessary for hydroxylation of cholesterol;

-         takes part in metabolism of adrenaline;

-         is necessary for the metabolism of mineral elements (Fe, Ca);

- accelerates the synthesis of glycogen in liver.

While at sea in May 1747, Lind provided some crewmembers with two oranges

and one lemon per day, in addition to normal rations, while others continued on cider, vinegar or seawater, along with their normal rations. In the history of science this is considered to be the first example of a controlled experiment comparing results on two populations of a factor applied to one group only with all other factors the same.

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In the hypovitaminosis of vitamin C the disease scurvy is developed. Main clinical symptoms of scurvy: delicacy, vertigo, palpitation, tachycardia, pain in the area of heart, dyspnea, petechias, odontorrhagia, dedentition.

Ascorbic acid and products of its decomposition are excreted from the organism via kidneys. In normal conditions 20-30 mg or 113,5-170,3 mkmol of ascorbic acid is excreted per day with urine.

In animal and plant tissues rather large concentrations of ascorbic acid are present, in comparison with other water-soluble vitamins; e.g., human blood plasma contains about 1 mg of ascorbic acid per 100 ml. Ascorbic acid is especially abundant in citrus fruits, tomatoes, currant, onion, garlic, cabbage, fruits of wild rose, needles of a pine-tree.

Sources of vitamin C

Vitamin C source

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Vitamin C is obtained through the diet by the vast majority of the world's population. The richest natural sources are fruits and vegetables, and of those, the camu camu fruit and the billygoat plum contain the highest concentration of the vitamin. It is also present in some cuts of meat, especially liver. Vitamin C as ascorbic acid is the most widely taken nutritional supplement and is available in a variety of forms from tablets and drink mixes to pure ascorbic acid crystals in capsules or as plain powder.

Plant sources

Rose hips are a particularly rich source of vitamin C Citrus fruits (orange, lemon, grapefruit, lime), tomatoes, and potatoes are good common sources of vitamin C. Other foods that are good sources of vitamin C include papaya, broccoli, brussels sprouts, black currants, strawberries, cauliflower, spinach, cantaloupe, kiwifruit, cranberries and red peppers. Ascorbic acid in food is largely destroyed by cooking.

Although the symptoms of scurvy in man can be prevented by as little as 20 mg of ascorbic acid per day, there are evidences that far larger amounts may be required for completely normal physiological function and well-being. Day necessity of vitain C: 50 - 70 mg. But in different diseases, pregnancy, in hard physical and mental work, in growing organism, after operations the day requirement of vitamin C increased.

Vitamin P (bioflavonoids).

This is the group of compounds (rutin, hesperedin, katecholamines) supporting the elasticity of capillaries, strengthen their walls and decrease the permeability.

Vitamin P takes part in the oxidative-reduction processes. It oppresses the activity of enzyme hyaluronidase protecting the hyaluronic acid which is necessary for elasticity of vessel walls.

The deficiency of vitamin P in organism results in the petechias (dot hemorrhages on skin).

Day necessity of vitamin P is not clear exactly (about 25-50 mg). In some diseases 1-2 g per day of vitamin P is administrated.