Biochemistry – subject and main tasks

Biochemistry – subject and main tasks. Investigation of structure and physical-chemical properties of proteins-enzymes. Mechanism of enzyme action, kinetic of enzymatic catalysis, role of cofactors and coenzyme vitamins in catalytic action of enzymes.

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.

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 (1837–1900) 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.

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.

http://www.youtube.com/watch?v=AFbPHlhI13g&feature=related

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

http://www.youtube.com/watch?v=KED6BHVM97s&feature=related

44

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.

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).

45a

http://www.youtube.com/watch?v=cXLpxe6sPwI&feature=related

 

Most enzymes are much larger than the substrates they act on, and only a very small portion of the enzyme (around 3–4 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.

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.

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.

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 denatured—that is, unfolded and inactivated—by 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.

47

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.

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.

 

48

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

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.

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.

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.

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.

 

EnzymePathControlRegulation of Enzyme Activity

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

 

 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.

http://www.youtube.com/watch?v=duN73LFWNlo&feature=related

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.

Å. 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.

 

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

 enzyme_substrate

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.

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 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.

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.

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.

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.

Feedback inhibition

 Enzyme Regulation

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.

 

 

 The principle of international classification and nomenclature of enzymes.

 

The first general principle of these 'Recommendations' is that names purporting to be names of enzymes, especially those ending in -ase, should be used only for single enzymes, i.e. single catalytic entities. They should not be applied to systems containing more than one enzyme. When it is desired to name such a system on the basis of the overall reaction catalysed by it, the word system should be included in the name. For example, the system catalysing the oxidation of succinate by molecular oxygen, consisting of succinate dehydrogenase, cytochrome oxidase, and several intermediate carriers, should not be named succinate oxidase, but it may be called the succinate oxidase system. Other examples of systems consisting of several structurally and functionally linked enzymes (and cofactors) are the pyruvate dehydrogenase system, the similar 2-oxoglutarate dehydrogenase system, and the fatty acid synthase system.

To classify an enzyme according to the type of reaction catalysed, it is occasionally necessary to choose between alternative ways of regarding a given reaction. In general, that alternative should be selected which fits in best with the general system of classification and reduces the number of exceptions.

 Common and Systematic Names

The first Enzyme Commission gave much thought to the question of a systematic and logical nomenclature for enzymes, and finally recommended that there should be two nomenclatures for enzymes, one systematic, and one working or trivial. The systematic name of an enzyme, formed in accordance with definite rules, showed the action of an enzyme as exactly as possible, thus identifying the enzyme precisely. The trivial name was sufficiently short for general use, but not necessarily very systematic; in a great many cases it was a name already in current use. The introduction of (often cumbersome) systematic names was strongly criticised. In many cases the reaction catalysed is not much longer than the systematic name and can serve just as well for identification, especially in conjunction with the code number.

The Commission for Revision of Enzyme Nomenclature discussed this problem at length, and a change in emphasis was made. It was decided to give the trivial names more prominence in the Enzyme List; they now follow immediately after the code number, and are described as Common Name. Also, in the index the common names are indicated by an asterisk. Nevertheless, it was decided to retain the systematic names as the basis for classification for the following reasons:

(i) the code number alone is only useful for identification of an enzyme when a copy of the Enzyme List is at hand, whereas the systematic name is self-explanatory;

(ii) the systematic name stresses the type of reaction, the reaction equation does not;

(iii) systematic names can be formed for new enzymes by the discoverer, by application of the rules, but code numbers should not be assigned by individuals;

(iv) common names for new enzymes are frequently formed as a condensed version of the systematic name; therefore, the systematic names are helpful in finding common names that are in accordance with the general pattern.

 Scheme for the classification of enzymes and the generation of EC numbers

The first Enzyme Commission, in its report in 1961, devised a system for classification of enzymes that also serves as a basis for assigning code numbers to them. These code numbers, prefixed by EC, which are now widely in use, contain four elements separated by points, with the following meaning:

(i) the first number shows to which of the six main divisions (classes) the enzyme belongs,

(ii) the second figure indicates the subclass,

(iii) the third figure gives the sub-subclass,

(iv) the fourth figure is the serial number of the enzyme in its sub-subclass.

 

Enzyme Classifications

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.

Hydrolases. Mechanism of action of esterases, peptidases, glycosidases. Examples.

 Hydrolases.

These enzymes catalyse the hydrolytic cleavage of C-O, C-N, C-C and some other bonds, including phosphoric anhydride bonds. Although the systematic name always includes hydrolase, the common name is, in many cases, formed by the name of the substrate with the suffix -ase. It is understood that the name of the substrate with this suffix means a hydrolytic enzyme.

A number of hydrolases acting on ester, glycosyl, peptide, amide or other bonds are known to catalyse not only hydrolytic removal of a particular group from their substrates, but likewise the transfer of this group to suitable acceptor molecules. In principle, all hydrolytic enzymes might be classified as transferases, since hydrolysis itself can be regarded as transfer of a specific group to water as the acceptor. Yet, in most cases, the reaction with water as the acceptor was discovered earlier and is considered as the main physiological function of the enzyme. This is why such enzymes are classified as hydrolases rather than as transferases.

Some hydrolases (especially some of the esterases and glycosidases) pose problems because they have a very wide specificity and it is not easy to decide if two preparations described by different authors (perhaps from different sources) have the same catalytic properties, or if they should be listed under separate entries. An example is vitamin A esterase (formerly EC 3.1.1.12, now believed to be identical with EC 3.1.1.1). To some extent the choice must be arbitrary; however, separate entries should be given only when the specificities are sufficiently different.

Another problem is that proteinases have 'esterolytic' action; they usually hydrolyse ester bonds in appropriate substrates even more rapidly than natural peptide bonds. In this case, classification among the peptide hydrolases is based on historical priority and presumed physiological function.

The second figure in the code number of the hydrolases indicates the nature of the bond hydrolysed; EC 3.1 are the esterases; EC 3.2 the glycosylases, and so on.

The third figure normally specifies the nature of the substrate, e.g. in the esterases the carboxylic ester hydrolases (EC 3.1.1), thiolester hydrolases (EC 3.1.2), phosphoric monoester hydrolases (EC 3.1.3); in the glycosylases the O-glycosidases (EC 3.2.1), N-glycosylases (EC 3.2.2), etc. Exceptionally, in the case of the peptidyl-peptide hydrolases the third figure is based on the catalytic mechanism as shown by active centre studies or the effect of pH.

Esterases - enzymes that hydrolyse esters, i.e. cleave the ester linkage to form free acid and alcohol. Those that hydrolyse the ester linkages of fats are generally known as lipases, and those that hydrolyse phospholipids as phospholipases. Any enzymes catalyses the hydrolysis of an ester.

Peptidase, also called protease or proteinase, is a type of enzyme that helps to break down proteins in the body. This type of enzyme occurs naturally in the living things and forms part of many metabolic processes. They form part of the larger systems in the body, including the digestive, immune, and blood circulation systems. These enzymes are classified into five different groups: aspartic proteinases, cysteine proteinases, metalloproteinases, serine proteinases, and threonine proteases.

In the digestive system, peptidases break down proteins by destroying the chains between their amino acids, and many can usually be found in the digestive tract. When protein enters the body, it needs to be digested and broken down into smaller molecules so that it can be used. This type of enzyme is responsible for this catabolic process.

Aspartic proteinases can usually be found in an acidic environment like the stomach. They are responsible for the breakdown of food and are also called pepsins. Other places that aspartic proteinases can be found are in blood plasma and in the immune system.

Glycoside hydrolases (also called glycosidases or glycosyl hydrolases) catalyze the hydrolysis of the glycosidic linkage to release smaller sugars. They are extremely common enzymes with roles in nature including degradation of biomass such as cellulose and hemicellulose, in anti-bacterial defense strategies (e.g., lysozyme), in pathogenesis mechanisms (e.g., viral neuraminidases) and in normal cellular function (e.g., trimming mannosidases involved in N-linked glycoprotein biosynthesis). Together with glycosyltransferases, glycosidases form the major catalytic machinery for the synthesis and breakage of glycosidic bonds.

The subclasses and sub-subclasses are formed according to principles indicated below. he main divisions and subclasses are:

Oxidoreductases

Oxidoreductases are a class of enzymes that catalyze oxidoreduction reactions.  Oxidoreductases catalyze the transfer of electrons from one molecule (the oxidant) to another molecule (the reductant).  Oxidoreductases catalyze reactions similar to the following, A + B → A + B where A is the oxidant and B is the reductant.  Oxidorecuctases can be oxidases or dehydrogenasesOxidases are enzymes involved when molecular oxygen acts as an acceptor of hydrogen or electrons.  Whereas, dehydrogenases are enzymes that oxidize a substrate by transferring hydrogen to an acceptor that is either NAD+/NADP+ or a flavin enzyme.  Other oxidoreductases include peroxidases, hydroxylases, oxygenases, and reductases. Peroxidases are localized in peroxisomes, and catalyzes the reduction of hydrogen peroxide. Hydroxylases add hydroxyl groups to its substrates.

 Oxygenases incorporate oxygen from molecular oxygen into organic substrates. Reductases catalyze reductions, in most cases reductases can act like an oxidases.

Oxidoreductase enzymes play an important role in both aerobic and anaerobic metabolism.  They can be found in glycolysis, TCA cycle, oxidative phosphorylation, and in amino acid metabolism.  In glycolysis, the enzyme glyceraldehydes-3-phosphate dehydrogenase catalyzes the reduction of NAD+ to NADH.  In order to maintain the re-dox state of the cell, this NADH must be re-oxidized to NAD+, which occurs in the oxidative phosphorylation pathway.  Additional NADH molecules are generated in the TCA cycle.  The product of glycolysis, pyruvate enters the TCA cycle in the form of acetyl-CoA.  During anaerobic glycolysis, the oxidation of NADH occurs through the reduction of pyruvate to lactate.  The lactate is then oxidized to pyruvate in muscle and liver cells, and the pyruvate is further oxidized in the TCA cycle.  All twenty of the amino acids, except leucine and lysine, can be degraded to TCA cycle intermediates.  This allows the carbon skeletons of the amino acids to be converted into oxaloacetate and subsequently into pyruvate.  The gluconeogenic pathway can then utilize the pyruvate forme

Transferases

Transferases are enzymes transferring a group, e.g. a methyl group or a glycosyl group, from one compound (generally regarded as donor) to another compound (generally regarded as acceptor). The systematic names are formed according to the scheme donor:acceptor grouptransferase. The common names are normally formed according to acceptor grouptransferase or donor grouptransferase. In many cases, the donor is a cofactor (coenzyme) charged with the group to be transferred. A special case is that of the transaminases (see below).

Some transferase reactions can be viewed in different ways. For example, the enzyme-catalysed reaction   X-Y + Z = X + Z-Y may be regarded either as a transfer of the group Y from X to Z, or as a breaking of the X-Y bond by the introduction of Z. Where Z represents phosphate or arsenate, the process is often spoken of as 'phosphorolysis' or 'arsenolysis', respectively, and a number of enzyme names based on the pattern of phosphorylase have come into use. These names are not suitable for a systematic nomenclature, because there is no reason to single out these particular enzymes from the other transferases, and it is better to regard them simply as Y-transferases.

In the above reaction, the group transferred is usually exchanged, at least formally, for hydrogen, so that the equation could more strictly be written as:

X-Y + Z-H = X-H + Z-Y

Another problem is posed in enzyme-catalysed transaminations, where the -NH2 group and -H are transferred to a compound containing a carbonyl group in exchange for the =O of that group, according to the general equation:

R1-CH(-NH2)-R2 + R3-CO-R4 [arrow right]R1-CO-R2 + R3-CH(-NH2)-R4

The reaction can be considered formally as oxidative deamination of the donor (e.g. amino acid) linked with reductive amination of the acceptor (e.g. oxo acid), and the transaminating enzymes (pyridoxal-phosphate proteins) might be classified as oxidoreductases. However, the unique distinctive feature of the reaction is the transfer of the amino group (by a well-established mechanism involving covalent substrate-coenzyme intermediates), which justified allocation of these enzymes among the transferases as a special subclass (EC 2.6.1, transaminases).

Lyases

Lyases are enzymes cleaving C-C, C-O, C-N, and other bonds by elimination, leaving double bonds or rings, or conversely adding groups to double bonds. The systematic name is formed according to the pattern substrate group-lyase. The hyphen is an important part of the name, and to avoid confusion should not be omitted, e.g. hydro-lyase not 'hydrolyase'. In the common names, expressions like decarboxylase, aldolase, dehydratase (in case of elimination of CO2, aldehyde, or water) are used. In cases where the reverse reaction is much more important, or the only one demonstrated, synthase (not synthetase) may be used in the name. Various subclasses of the lyases include pyridoxal-phosphate enzymes that catalyse the elimination of a β- or γ-substituent from an α-amino acid followed by a replacement of this substituent by some other group. In the overall replacement reaction, no unsaturated end-product is formed; therefore, these enzymes might formally be classified as alkyl-transferases (EC 2.5.1...). However, there is ample evidence that the replacement is a two-step reaction involving the transient formation of enzyme-bound α,β(or β,γ)-unsaturated amino acids. According to the rule that the first reaction is indicative for classification, these enzymes are correctly classified as lyases. Examples are tryptophan synthase (EC 4.2.1.20) and cystathionine β-synthase (EC 4.2.1.22).

The second figure in the code number indicates the bond broken: EC 4.1 are carbon-carbon lyases, EC 4.2 carbon-oxygen lyases and so on.

The third figure gives further information on the group eliminated (e.g. CO2 in EC 4.1.1, H2O in EC 4.2.1).

Isomerases

These enzymes catalyse geometric or structural changes within one molecule. According to the type of isomerism, they may be called racemases, epimerases, cis-trans-isomerases, isomerases, tautomerases, mutases or cycloisomerases.

In some cases, the interconversion in the substrate is brought about by an intramolecular oxidoreduction (EC 5.3); since hydrogen donor and acceptor are the same molecule, and no oxidized product appears, they are not classified as oxidoreductases, even though they may contain firmly bound NAD(P)+.

The subclasses are formed according to the type of isomerism, the sub-subclasses to the type of substrates.

Ligases

Ligases are enzymes catalysing the joining together of two molecules coupled with the hydrolysis of a diphosphate bond in ATP or a similar triphosphate. The systematic names are formed on the system X:Y ligase (ADP-forming). In earlier editions of the list the term synthetase has been used for the common names. Many authors have been confused by the use of the terms synthetase (used only for Group 6) and synthase (used throughout the list when it is desired to emphasis the synthetic nature of the reaction).

Consequently NC-IUB decided in 1983 to abandon the use of synthetase for common names, and to replace them with names of the type X-Y ligase. In a few cases in Group 6, where the reaction is more complex or there is a common name for the product, a synthase name is used (e.g. EC 6.3.2.11 and EC 6.3.5.1).

It is recommended that if the term synthetase is used by authors, it should continue to be restricted to the ligase group.

The second figure in the code number indicates the bond formed: EC 6.1 for C-O bonds (enzymes acylating tRNA), EC 6.2 for C-S bonds (acyl-CoA derivatives), etc. Sub-subclasses are only in use in the C-N ligases.

In a few cases it is necessary to use the word other in the description of subclasses and sub-subclasses. They have been provisionally given the figure 99, in order to leave space for new subdivisions.

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

 

Enzymes, specific to different organs. Localization of enzymes in cell’s organells

 

An adaptive enzyme or inducible enzyme is an enzyme that is expressed only under conditions in which it is clear of adaptive value, as opposed to a constitutive enzyme which is produced all the time. The Inducible enzyme is used for the breaking-down of things in the cell. It is also a part of the Operon Model, which illustrates a way for genes to turn "on" and "off". The Inducer causes the gene to turn on (controlled by the amount of reactant which turns the gene on). Then there's the repressor protein that turns genes off.

The inducer can remove this repressor, turning genes back on. The operator is a section of DNA where the repressor binds to shut off certain genes; the promoter is the section of DNA where the RNA polymerase binds. Lastly, the regulatory gene is the gene for the repressor protein. An example of inducible enzyme is COX-2 which is synthesized in macrophages to produce Prostaglandin E2 while the constitutive enzyme COX-1 (another isozyme in COX family) is always produced in variety of organisms in body (like stomach).

Constitutive enzymes are produced constitutively by the cell under all physiological conditions. Therefore, they are not controlled by induction or repression.

Constitutive enzymes are produced in constant amounts without regard to the physiological demand or the concentration of the substrate. They are continuously synthesized because their role in maintaining cell processes or structure is indispensable.

 

The methods of separation and purification of enzymes

 

Analysis of the biological properties Understand its structure.

Study interactions. No single procedure can be used to isolate every protein

Exploit specific characteristics (structure or function) of the protein. Different steps should exploit a different characteristic. Ensure method has little/no effect on function.

Ammonium sulphate precipitation (40%) exploits changes in the solubility of proteins as consequence of a change in ionic strength (salt conc.) of the solution

At low salt, the solubility of a protein increases with salt concentration,

‘SALTING IN’

 

But as salt conc. (ionic strength) is increased further, the solubility of the protein begins to decrease, until a point where the protein is precipitated from solution,

‘SALTING OUT’

 Ion Exchange Chromatography (IEC)

Separates molecules based on their charge.

The side-chain groups of some amino acids are ionizable, e.g., lysine, arginine, histidine, glutamic acid, aspartic acid as are the N-terminal amino and C-terminal carboxyl groups. Thus proteins are charge molecule s and can have a different charge at a given pH because they have different compositions of ionizable amino acids.

For any given amphoteric protein, there will be a pH at which its overall charge is 0

(No. of negative charges equals the No. of positive charges).

This is referred to as the ISOELECTRIC POINT (pI) or ISOTONIC POINT of the protein. At a pH above its pI a protein will have a net negative charge while.

At a pH below its pI a protein will have a net positive charge.

 

Gel Filtration Chromatography (GFC)

GFC (also Size Exclusion Chromatography, Molecular Sieve Chromatography or Molecular Exclusion Chromatography)

Separates molecules based on their size (& shape)

It can also be used to determine the size and molecular weight of a protein

Separation occurs due to the differential diffusion of various molecules into gel pores in a porous matrix. For protein purification, the matrix typically consists of porous beads (with pores of a specific size distribution) of an inert, highly hydrated gel.

 

Affinity Chromatography:

Separates molecules based on specific interactions between the protein of interest and the column matrix E.g. Antibodies which bind Protein. Enzyme which binds a co-enzyme or inhibitor.

A ligand is covalently bound to a solid matrix (usually agarose) which is then packed into a chromatography column When a mixture containing the protein of interest is applied to the column, the desired protein is bound by the immobilised

ligands, while all other proteins in the mixture, which should have no affinity for the ligand pass through and are discarded

Affinity chromatography (with HIS-tagged proteins).

Affinity chromatography can be performed using a number of  different protein tags. poly-hisitidine.

The histidine tag is very short (6 His residues).

Should not alter the conformation of the tagged protein.

Should not be involved in artificial interactions.

The poly-his tag binds to a nickel chelate resin.

Eluted by 1.0 M imidazole.

 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).

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.

Catalase

         Most catalases exist as tetramers of 60 or 75 kDa, each subunit containing an active site haem group buried deep within the structure, but which is accessible from the surface through hydrophobic channels.  The very rigid, stable structure of catalases is resistant to unfolding, which makes them uniquely stable enzymes that are more resistant to pH, thermal denaturation and proteolysis than most other enzymes.  Their stability and resistance to proteolysis is an evolutionary advantage, especially since they are produced during the stationary phase of cell growth when levels of proteases are high and there is a rapid rate of protein turnover.

Haem-containing catalases break down hydrogen peroxide by a two-stage mechanism in which hydrogen peroxide alternately oxidises and reduces the haem iron at the active site.  In the first step, one hydrogen peroxide molecule oxidises the haem to an oxyferryl species.  In the second step, a second hydrogen peroxide molecule is used as a reductant to regenerate the enzyme, producing water and oxygen.  Some catalases contain NADPH as a cofactor, which functions to prevent the formation of an inactive compound.  Catalases may have another role: the generation of ROS, possibly hydroperoxides, upon UVB irradiation.  In this way, UVB light can be detoxified through the generation of hydrogen peroxide, which can then be degraded by the catalase.  NADPH may play a role in providing the electrons needed to reduce molecular oxygen in the production of ROS.

Much of the hydrogen peroxide that is produced during oxidative cellular metabolism comes from the breakdown of one of the most damaging ROS, namely the superoxide anion radical (O2-).  Superoxide is broken down by superoxide dismutases into hydrogen peroxide and oxygen.  Superoxide is so damaging to cells that mutations in the superoxide dismutase enzyme can lead to ALS, which is characterised by the loss of motoneurons in the spinal cord and brain stem, possibly involving the activation of caspase-12 and the apoptosis cascade via oxidative stress. 

Regulation of Antioxidant Enzymes

         Antioxidant enzymes, including catalase, form the first line of defence against free radicals, therefore their regulation depends mainly upon the oxidant status of the cell. 

However, there are other factors involved in their regulation, including the enzyme-modulating action of various hormones such as growth hormone, prolactin and melatonin.  Melatonin is a derivative of the amino acid tryptophan that acts as a neurohormone in mammals, but is also synthesized by many other species, including plants, algae and bacteria.  Melatonin has been shown to markedly protect both membrane lipids and nuclear DNA from oxidative damage.  Melatonin can directly neutralise several ROS, including hydrogen peroxide.  It can also stimulate various antioxidant enzymes, including catalase, either by increasing their activity or by stimulating gene expression for these enzymes.  The decrease in melatonin levels observed with age correlates with an increase in neurogenerative disorders such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and stroke, all of which may involve oxidative stress.  In general, the production of ROS increases with aging and is associated with DNA damage to the tissues.

         By contrast, growth hormone, and possibly prolactin, was found to decrease catalase and other antioxidant enzymes in various tissues in mice, suggesting that this hormone acts as a suppressor of key antioxidant components.

 

The origin of blood enzymes

Do you mean a blood enzymes test, or more generally, enzymes in the blood?

Enzymes are proteins that carry out chemical reactions (as opposed to structural enzymes). Most of the detectable enzymes in the blood come from the various tissues and organs of the body. Abnormal levels may reflect problems with a particular organ.

The most common blood enzymes test is for liver enzymes. When the cells of the liver are damaged, such as from a viral infection, their enzymes can leak out and be detected in the blood. Another common test measures enzymes from heart damage, such as from a heart attack.

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.

 

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.

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.

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 îr 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 0°C 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.