STRUCTURE OF PROTEINS, METHODS OF ITS DETERMINATION. PHYSICAL-CHEMICAL PROPERTIES OF SIMPLE AND COMPLEX
PROTEINS, PRECIPITATION REACTIONS. STUDING OF THE STRUCTURE AND
PHYSICAL-CHEMICAL PROPERTIES OF ENZYMES. MECHANISM OF ENZYME ACTION. VITAMINS
AS NUTRITION COMPONENTS: EXOGENIC AND ENDOGENIC HYPOVITAMINOSIS. WATER AND FAT
SOLUBLE VITAMINS.
Biochemistry is the study of the chemical processes and transformations in living organisms. It deals with the structure and function of cellular components, such as proteins, carbohydrates, lipids, nucleic acids, and other biomolecules. Chemical biology aims to answer many questions arising from
biochemistry by using tools developed within synthetic chemistry.
Although there are a vast number of
different biomolecules, many are complex and large molecules (called polymers) that are composed of similar repeating
subunits (called monomers). Each class of
polymeric biomolecule has a different set of subunit types. For example, a
protein is a polymer made up of many amino acids. Biochemistry studies the chemical
properties of important biological molecules, like proteins, in particular the
chemistry of enzyme-catalyzed reactions (enzymes are a type of protein).
The biochemistry of
cell metabolism and the endocrine system has been extensively described. Other areas
of biochemistry include the genetic code (DNA, RNA), protein synthesis, cell membrane transport, and signal transduction.
This article only discusses terrestrial biochemistry (carbon- and water-based), as all the life forms we know are on Earth. Since life forms alive today are hypothesized by most to have
descended from the same common ancestor, they would naturally have similar biochemistries, even for matters
that seem to be essentially arbitrary, such as handedness of various biomolecules. It is unknown whether alternative
biochemistries are possible or
practical.
History of biochemistry
In the 19th century, when studying
the fermentation of sugar to alcohol by yeast, Louis Pasteur came to the
conclusion that this fermentation was catalyzed by a vital force contained
within the yeast cells called "ferments", which were thought to
function only within living organisms. He wrote that "alcoholic
fermentation is an act correlated with the life and organization of the yeast
cells, not with the death or putrefaction of the cells."
In 1878 German physiologist Wilhelm
Kühne (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
Having shown that enzymes could
function outside a living cell, the next step was to determine their
biochemical nature. Many early workers noted that enzymatic activity was
associated with proteins, but several scientists (such as Nobel laureate
Richard Willstätter) argued that proteins were merely carriers for the
true enzymes and that proteins per se were incapable of catalysis. However, in
1926, James B. Sumner showed that the enzyme urease was a pure protein and
crystallized it; Sumner did likewise for the enzyme catalase in 1937. The
conclusion that pure proteins can be enzymes was definitively proved by
Northrop and Stanley, who worked on the digestive enzymes pepsin (1930),
trypsin and chymotrypsin. These three scientists were awarded the 1946 Nobel
Prize in Chemistry.
This discovery that enzymes could be
crystallized eventually allowed their structures to be solved by x-ray
crystallography. This was first done for lysozyme, an enzyme found in tears,
saliva and egg whites that digests the coating of some bacteria; the structure
was solved by a group led by David Chilton Phillips and published in 1965. This
high-resolution structure of lysozyme marked the beginning of the field of
structural biology and the effort to understand how enzymes work at an atomic
level of detail.
Originally, it was generally believed that
life was not subject to the laws of science the way non-life was. It was
thought that only living beings could produce the molecules of life (from
other, previously existing biomolecules). Then,
in 1828, Friedrich Wöhler published a paper about the synthesis of urea, proving that organic compounds can be created artificially. The
dawn of biochemistry may have been the discovery of the first enzyme, diastase (today called amylase), in 1833 by Anselme Payen. Eduard Buchner contributed the first demonstration of a
complex biochemical process outside of a cell in 1896: alcoholic fermentation
in cell extracts of yeast. Although the term “biochemistry” seems to have been
first used in 1882, it is generally accepted that the formal coinage of
biochemistry occurred in 1903 by Carl Neuber, a German chemist. Since then, biochemistry has advanced,
especially since the mid-20th century, with the development of new techniques such
as chromatography, X-ray diffraction, NMR spectroscopy, radioisotopic labelling, electron microscopy and molecular dynamics simulations. These techniques allowed for the
discovery and detailed analysis of many molecules and metabolic pathways of the cell, such as glycolysis and the Krebs cycle (citric acid cycle).
Today, the findings of biochemistry are
used in many areas, from genetics to molecular biology and from agriculture to medicine.
Characteristics
of Enzymes
Enzymes
are proteins that catalyze (i.e. accelerate) chemical reactions. In these
reactions, the molecules at the beginning of the process are called substrates,
and the enzyme converts them into different molecules, the products. Almost all
processes in a biological cell need enzymes in order to occur at significant
rates. Since enzymes are extremely selective for their substrates and speed up
only a few reactions from among many
possibilities, the set of enzymes made in a cell determines which metabolic
pathways occur in that cell.
Like all catalysts, enzymes work by
lowering the activation energy (ΔG‡) for a reaction, thus dramatically accelerating the
rate of the reaction. Most enzyme reaction rates are millions of times faster
than those of comparable uncatalyzed reactions. As with all catalysts, enzymes
are not consumed by the reactions they catalyze, nor do they alter the equilibrium
of these reactions. However, enzymes do differ from most
other catalysts by being much more specific. Enzymes are known to catalyze
about 4,000 biochemical reactions. Not all biochemical catalysts are proteins,
since some RNA molecules called ribozymes also catalyze reactions.
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
Enzymes are protein molecules that are tailored to recognize and bind specific
reactants and speed their conversion into products. These proteins are
responsible for increasing the rates of all of the many thousand of reaction
taking place inside cells.
All enzymatic proteins have several characteristics in common table 1.
Table 1: Characteristics of enzymes proteins
1. |
Enzymes combine briefly
with reactants during an enzyme-catalyzed reaction. |
2. |
Enzymes are released
unchanged after catalyzing the conversion of reactants to Product |
3. |
Enzymes are specific in
their activity; each enzyme catalyzes the reaction of a single type of
molecules or a group of closely related molecules. |
4. |
Enzymes are saturated
by high substrate concentrations. |
5. |
Many enzymes contain nonproteins
groups called cofactors, which contribute to their activity. Inorganic
cofactors are all metallic ions. Organic cofactors, called coenzymes, are
complex groups derived from vitamins. |
6. |
Many enzymes are pH and
temperature sensitive |
The rate of
combination and release, known as the turnover number, lies near 1000 per
second for most enzymes. Some enzymes have turnover numbers as small as 100 per
second or as large as 10 million per second. As a result of enzyme turnover, a
relatively small number of enzyme molecules can catalyze a large number of
reactant molecules.
The part of an enzyme that combines with substrate molecule is the active
site. In most enzymes the active site is located in a cavity or pocket on the
enzyme surface, frequently within a cleft marking the boundary between two or
more major domains. Within the cleft or pocket, amino acid side groups are
situated to fit and bind parts of substrate molecules that are critical to the
reaction catalyzed by the enzyme. The active site also separates substrate
molecules from the surrounding solutions and place them in environments with
unique characteristics, including partial or complete exclusion of water.
How Enzymes Lower the Energy of Activation
The mechanisms by which enzymes lower the energy of
activation are still not totally understood. However, the mechanisms are
believed to be directly or indirectly related to achievement of what is known
as the transition state for a reaction. During any chemical interaction the
reactants briefly enter a state in which old chemical bonds are incompletely
broken and new ones are incompletely formed. In this transition state electron
orbital assume intermediate positions between their locations in the reactants
and their positions in the products. The transition state is highly unstable
and can easily move in either direction with little change in energy - forward
toward products ore back ward toward reactants. In effect, achievement of the
transition state places a reacting system in a poised and precariously balanced
position at the top of the activation energy barrier.
For example, in the
transfer of a phosphate group from one molecule to another, a transition state
is set up in which both molecules (shown as X and Y in Figure 2) link to the
phosphate group a fraction of a second via transitory bonds (dotted lines).
This unstable state can change readily in the direction of either products or
unchanged reactants.
Enzymes
as Biological Catalysts
In
cells and organisms most reactions are catalyzed by enzymes, which are
regenerated during the course of a reaction. These biological catalysts are
physiologically important because they speed up the rates of reactions that
would otherwise be too slow to support life. Enzymes increase reaction rates---
sometimes by as much as one millionfold, but more typically by about one
thousand fold. Catalysts speed up the forward and reverse reactions
proportionately so that, although the magnitude of the rate constants of the
forward and reverse reactions is are increased, the ratio of the rate constants
remains the same in the presence or absence of enzyme. Since the equilibrium
constant is equal to a ratio of rate constants, it is apparent that enzymes and
other catalysts have no effect on the equilibrium constant of the reactions
they catalyze.
Enzymes increase reaction rates by decreasing the
amount of energy required to form a complex of reactants that is competent to
produce reaction products. This complex is known as the activated state or
transition state complex for the reaction. Enzymes and other catalysts
accelerate reactions by lowering the energy of the transition state. The free
energy required to form an activated complex is much lower in the catalyzed
reaction. The amount of energy required to achieve the
transition state is lowered; consequently, at any instant a greater proportion
of the molecules in the population can achieve the transition state. The result
is that the reaction rate is increased.
A number of
mechanisms operate to contribute to formation of the transition state. One is
bringing reacting molecules into close proximity. Many reactions involve
combination or interaction of two or more reactant molecules. For the reaction
to take place, the substrate molecule must collide. The required collisions may
be rate among reactant molecules suspended in free solution, particularly if
the substrates are present in low concentrations. Binding at the active site of
an enzyme brings the reactants close together, raising their effective concentration
in the active site to many time the concentration in the surrounding solution.
A second contribution mechanism is orienting reactants in positions
favoring their interaction. Binding at the active site may bring substrate molecules
into an arrangement in which they can collide ate exactly the correct positions
and angles required for achievement of the transition state.
The third contributing mechanism is exposing reactant molecules to
altered environments that promote their interaction. Enzyme-Substrate
Interactions
The
favored model of enzyme substrate interaction is known as the induced fit
model. This model proposes that the initial interaction between enzyme and
substrate is relatively weak, but that these weak interactions rapidly induce
conformational changes in the enzyme that strengthen binding and bring
catalytic sites close to substrate bonds to be altered. After binding takes
place, one or more mechanisms of catalysis generates transition- state
complexes and reaction products. The possible mechanisms of catalysis are four
in number:
1.
Catalysis by Bond Strain: In this form of catalysis, the induced structural
rearrangements that take place with the binding of substrate and enzyme
ultimately produce strained substrate bonds, which more easily attain the
transition state. The new conformation often forces substrate atoms and bulky
catalytic groups, such as aspartate and glutamate, into conformations that
strain existing substrate bonds.
2. Catalysis by Proximity and Orientation:
Enzyme-substrate interactions orient reactive groups and bring them into
proximity with one another. In addition to inducing strain, groups such as
aspartate are frequently chemically reactive as well, and their proximity and
orientation toward the substrate thus favors their participation in catalysis.
3.
Catalysis Involving Proton Donors (Acids) and Acceptors (Bases): Other
mechanisms also contribute significantly to the completion of catalytic events
initiated by a strain mechanism, for example, the use of glutamate as a general
acid catalyst (proton donor).
4.
Covalent Catalysis: In catalysis that takes place by covalent mechanisms, the
substrate is oriented to active sites on the enzymes in such a way that a
covalent intermediate forms between the enzyme or coenzyme and the substrate.
One of the best-known examples of this mechanism is that involving proteolysis
by serine proteases, which include both digestive enzymes (trypsin,
chymotrypsin, and elastase) and several enzymes of the blood clotting cascade.
These proteases contain an active site serine whose R group hydroxyl forms a
covalent bond with a carbonyl carbon of a peptide bond, thereby causing
hydrolysis of the peptide bond.
Some reactions, for
example, take place more readily in nonpolar environments. Active sites may
create such an environment by binding reactants so closely that water molecules
are excluded. Another important environmental change is creation of acidic or
basic conditions by groups in the active site that release or take up H+
.
Enzyme activity can be affected by other molecules. Inhibitors are
molecules that decrease enzyme activity; activators are molecules that increase
activity. Many drugs and poisons are enzyme inhibitors. Activity is also
affected by temperature, pH, and the concentration of substrate. Some enzymes
are used commercially, for example, in the synthesis of antibiotics. In
addition, some household products use enzymes to speed up biochemical reactions
(e.g., enzymes in biological washing powders break down protein or fat stains
on clothes; enzymes in meat tenderizers break down proteins, making the meat
easier to chew).
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.
Structures and mechanisms
Ribbon-diagram showing carbonic anhydrase II. The grey sphere is the
zinc cofactor in the active site. Diagram drawn from PDB 1MOO.The activities of
enzymes are determined by their three-dimensional structure.
This binding can serve to increase or decrease the enzyme's activity,
providing a means for feedback regulation. Enzymes
are catalysts. Most are proteins. (A few ribonucleoprotein enzymes
have been discovered and, for some of these, the catalytic activity is in the
RNA part rather than the protein part. Link to discussion of these ribozymes.)
Enzymes bind temporarily to one or more of the reactants of the reaction they catalyze. In doing so, they lower the amount of
activation energy needed and thus speed up the reaction.
|
Examples:
· Catalase. It catalyzes the decomposition of hydrogen peroxide into water and
oxygen.
2H2O2
-> 2H2O + O2
One molecule of
catalase can break 40 million molecules of hydrogen peroxide each second.
· Carbonic anhydrase. It is found in red blood cells where it
catalyzes the reaction
CO2 + H2O <-> H2CO3
It enables red blood cells to transport carbon dioxide from the tissues to
the lungs. One molecule of carbonic anhydrase can process one million molecules
of CO2 each second.
·
Acetylcholinesterase. It catalyzes the breakdown of the neurotransmitter acetylcholine
at several types of synapses as well as at the
neuromuscular junction -
the specialized synapse that triggers the contraction of skeletal muscle.
One molecule of
acetylcholinesterase breaks down 25,000 molecules of acetylcholine each second.
This speed makes possible the rapid "resetting" of the synapse for
transmission of another nerve impulse.
Like all proteins, enzymes are made as long, linear chains of amino
acids that fold to produce a three-dimensional product. Each unique amino acid
sequence produces a unique structure, which has unique properties. Individual
protein chains may sometimes group together to form a protein complex. Most
enzymes can be 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.
Some of the enzymes showing the highest specificity and accuracy are
involved in the copying and expression of the genome. These enzymes have
"proof-reading" mechanisms. Here, an enzyme such as DNA polymerase
catalyses a reaction in a first step and then checks that the product is
correct in a second step. This two-step process results in average error rates
of less than 1 error in 100 million reactions in high-fidelity mammalian
polymerases. Similar proofreading mechanisms are also found in RNA polymerase,
aminoacyl tRNA synthetases and ribosomes.
Some enzymes that produce secondary metabolites are described as
promiscuous, as they can act on a relatively broad range of different
substrates. It has been suggested that this broad substrate specificity is
important for the evolution of new biosynthetic pathways.
"Lock and key" model
Enzymes are very specific, and it was suggested by Emil Fischer in 1894
that this was because both the enzyme and the substrate possess specific
complementary geometric shapes that fit exactly into one another. This is often
referred to as "the lock and key" model. However, while this model
explains enzyme specificity, it fails to explain the stabilization of the
transition state that enzymes achieve.
Induced fit model
Diagrams to show the induced fit hypothesis of enzyme action.In 1958
Daniel Koshland suggested a modification to the lock and key model: since
enzymes are rather flexible structures, the active site can be reshaped by
interactions with the substrate as the substrate interacts with the enzyme. As
a result, the substrate does not simply bind to a rigid active site, the amino
acid side chains which make up the active site are molded into the precise
positions that enable the enzyme to perform its catalytic function. In some
cases, such as glycosidases, the substrate molecule also changes shape slightly
as it enters the active site.
nzymes can act in several ways, all of which lower ΔG‡:
Lowering the activation energy by creating an environment in which the
transition state is stabilized (e.g. straining the shape of a substrate - by
binding the transition-state conformation of the substrate/product molecules,
the enzyme distorts the bound substrate(s) into their transition state form,
thereby reducing the amount of energy required to complete the transition).
Providing an alternative pathway (e.g. temporarily reacting with the
substrate to form an intermediate ES Complex which would be impossible in the
absence of the enzyme).
Reducing the reaction entropy change by bringing substrates together in
the correct orientation to react. Considering ΔH‡
alone overlooks this effect.
Dynamics
and function
Recent investigations have provided new
insights into the connection between internal dynamics of enzymes and their
mechanism of catalysis. An enzyme's internal dynamics are described as
the movement of internal parts (e.g. amino acids, a group of amino acids, a
loop region, an alpha helix, neighboring beta-sheets or even entire domain) of
these biomolecules, which can occur at various time-scales ranging from femtoseconds
to seconds. Networks of protein residues throughout an enzyme's structure can
contribute to catalysis through dynamic motions. Protein
motions are vital to many enzymes, but whether small and fast vibrations or
larger and slower conformational movements are more important depends on the
type of reaction involved. These new insights also
have implications in understanding allosteric effects, producing designer
enzymes and developing new drugs.
Cofactors and coenzymes
Role
of Coenzymes
The
functional role of coenzymes is to act as transporters of chemical groups from
one reactant to another. The chemical groups carried can be as simple as the
hydride ion (H+ + 2e-) carried by NAD or the mole of hydrogen carried by FAD;
or they can be even more complex than the amine (-NH2) carried by pyridoxal
phosphate.
Since
coenzymes are chemically changed as a consequence of enzyme action, it is often
useful to consider coenzymes to be a special class of substrates, or second
substrates, which are common to many different holoenzymes. In all cases, the
coenzymes donate the carried chemical grouping to an acceptor molecule and are
thus regenerated to their original form. This regeneration of coenzyme and
holoenzyme fulfills the definition of an enzyme as a chemical catalyst, since
(unlike the usual substrates, which are used up during the course of a
reaction) coenzymes are generally regenerated.
Enzyme
Relative to Substrate Type
Although
enzymes are highly specific for the kind of reaction they catalyze, the same is
not always true of substrates they attack. For example, while succinic
dehydrogenase (SDH) always catalyzes an oxidation-reduction reaction and its
substrate is invariably succinic acid, alcohol dehydrogenase (ADH) always
catalyzes oxidation-reduction reactions but attacks a number of different
alcohols, ranging from methanol to butanol. Generally, enzymes having broad
substrate specificity are most active against one particular substrate. In the
case of ADH, ethanol is the preferred substrate.
Enzymes
also are generally specific for a particular steric configuration (optical
isomer) of a substrate. Enzymes that attack D sugars will not attack the
corresponding L isomer. Enzymes that act on L amino acids will not employ the
corresponding D optical isomer as a substrate. The enzymes known as racemases
provide a striking exception to these generalities; in fact, the role of
racemases is to convert D isomers to L isomers and vice versa. Thus racemases
attack both D and L forms of their substrate.
As
enzymes have a more or less broad range of substrate specificity, it follows
that a given substrate may be acted on by a number of different enzymes, each
of which uses the same substrate(s) and produces the same product(s). The
individual members of a set of enzymes sharing such characteristics are known
as isozymes. These are the products of genes that vary only slightly; often,
various isozymes of a group are expressed in different tissues of the body. The best studied set of isozymes is the lactate
dehydrogenase (LDH) system. LDH is a tetrameric enzyme composed of all possible
arrangements of two different protein subunits; the subunits are known as H
(for heart) and M (for skeletal muscle). These subunits combine in various
combinations leading to 5 distinct isozymes. The all H isozyme is
characteristic of that from heart tissue, and the all M isozyme is typically
found in skeletal muscle and liver. These isozymes all catalyze the same
chemical reaction, but they exhibit differing degrees of efficiency. The
detection of specific LDH isozymes in the blood is highly diagnostic of tissue
damage such as occurs during cardiac infarct.
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
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.
Lysozyme: a model of enzyme action
A number of lysozymes are found in nature; in human tears and egg white,
for examples. The enzyme is antibacterial because it degrades the
polysaccharide that is found in the cell walls of many bacteria. It does this
by catalyzing the insertion of a water molecule at the position indicated by
the red arrow. This hydrolysis breaks the chain at that point.
The bacterial polysaccharide consists of long chains of alternating
amino sugars:
· N-acetylglucosamine (NAG)
· N-acetylmuramic acid (NAM)
These hexose units resemble glucose except for the presence of the side
chains containing amino groups.
Lysozyme is a globular protein with a deep cleft across part of its
surface. Six hexoses of the substrate fit into this cleft.
· With so many oxygen atoms in sugars, as many as 14 hydrogen
bonds form between the six amino sugars
and certain amino acid R
groups such as Arg-114, Asn-37,
Asn-44, Trp-62, Trp-63, and Asp-101.
· Some hydrogen bonds also form with the C=O groups of several peptide
bonds.
· In addition, hydrophobic interactions may help hold the substrate in
position.
X-ray crystallography
has shown that as lysozyme and its substrate unite, each is slightly deformed.
The fourth hexose in the chain (ring #4) becomes twisted out of its normal
position. This imposes a strain on the C-O bond on the ring-4 side of the oxygen
bridge between rings 4 and 5. It is just at this point that the polysaccharide
is broken. A molecule of water is inserted between these two hexoses, which
breaks the chain. Here, then, is a structural view of what it means to lower
activation energy. The energy needed to break this covalent bond is lower now
that the atoms connected by the bond have been distorted from their normal
position.
As for lysozyme itself, binding of the substrate induces a small
(~0.75Å) movement of certain amino acid residues so the cleft closes
slightly over its substrate. So the "lock" as well as the
"key" changes shape as the two are brought together. (This is
sometimes called "induced fit".)
The amino acid residues in the vicinity of rings 4 and 5 provide a
plausible mechanism for completing the catalytic act. Residue 35, glutamic acid
(Glu-35), is about 3Å from the -O- bridge that is to be broken.
The free carboxyl group of glutamic acid is a hydrogen ion donor and available
to transfer H+ to the oxygen atom. This would break the
already-strained bond between the oxygen atom and the carbon atom of ring 4.
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.
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.
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.
Several mechanisms work to make enzyme activity within the cell
efficient and well-coordinated.
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.
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.
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.
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..
Thermodynamics
Main aticles: Activation
energy, Thermodynamic equilibrium, and Chemical equilibrium
Diagram of a catalytic reaction, showing the
energy niveau at each stage of the reaction. The substrates usually need a
large amount of energy to reach the transition state, which then decays into the
end product. The enzyme stabilizes the transition state, reducing the energy
needed to form this species and thus reducing the energy required to form
products.As all catalysts, enzymes do not alter the position of the chemical
equilibrium of the reaction. Usually, in the presence
of an enzyme, the reaction runs in the same direction as it would without the
enzyme, just more quickly. However, in the absence of the enzyme, other
possible uncatalyzed, "spontaneous" reactions might lead to different
products, because in those conditions this different product is formed faster.
Furthermore, enzymes can couple two or more reactions, so that a
thermodynamically favorable reaction can be used to "drive" a
thermodynamically unfavorable one. For example, the hydrolysis of ATP is often
used to drive other chemical reactions.
Enzymes catalyze the forward and backward reactions equally. They do not
alter the equilibrium itself, but only the speed at which it is reached. For
example, carbonic anhydrase catalyzes its reaction in either direction
depending on the concentration of its reactants.
(in tissues; high CO2
concentration)
(in lungs; low CO2 concentration)
Nevertheless, if the equilibrium is greatly displaced in one direction,
that is, in a very exergonic reaction, the reaction is effectively
irreversible. Under these conditions the enzyme will, in fact, only catalyze
the reaction in the thermodynamically allowed direction.
Kinetics
Mechanism for a single substrate enzyme catalyzed reaction. The enzyme (E)
binds a substrate (S) and produces a product (P).Enzyme kinetics is the
investigation of how enzymes bind substrates and turn them into products. The
rate data used in kinetic analyses are obtained from enzyme assays. In 1913
Leonor Michaelis and Maud Menten proposed a quantitative theory of enzyme
kinetics, which is referred to as Michaelis-Menten kinetics. Their work was
further developed by G. E. Briggs and J. B. S. Haldane, who derived kinetic
equations that are still widely used today.
Michaelis-Menton Kinetics
In
typical enzyme-catalyzed reactions, reactant and product concentrations are
usually hundreds or thousands of times greater than the enzyme concentration.
Consequently, each enzyme molecule catalyzes the conversion to product of many
reactant molecules. In biochemical reactions, reactants are commonly known as
substrates. The catalytic event that converts substrate to product involves the
formation of a transition state, and it occurs most easily at a specific
binding site on the enzyme. This site, called the catalytic site of the enzyme,
has been evolutionarily structured to provide specific, high-affinity binding
of substrate(s) and to provide an environment that favors the catalytic events.
The complex that forms, when substrate(s) and enzyme combine, is called the
enzyme substrate (ES) complex. Reaction products arise when the ES complex
breaks down releasing free enzyme.
Between
the binding of substrate to enzyme, and the reappearance of free enzyme and
product, a series of complex events must take place. At a minimum an ES complex
must be formed; this complex must pass to the transition state (ES*); and the
transition state complex must advance to an enzyme product complex (EP). The
latter is finally competent to dissociate to product and free enzyme. The
series of events can be shown thus:
E
+ S <---> ES <---> ES* <---> EP <---> E + P
The kinetics of simple reactions like that above were
first characterized by biochemists Michaelis and Menten. The concepts
underlying their analysis of enzyme kinetics continue to provide the
cornerstone for understanding metabolism today, and for the development and
clinical use of drugs aimed at selectively altering rate constants and
interfering with the progress of disease states. The Michaelis-Menten equation
is a quantitative description of the relationship among the rate of an enzyme-
catalyzed reaction [v1], the concentration of substrate [S] and two constants,
Vmax and Km (which are set by the particular equation). The symbols used in the
Michaelis-Menton equation refer to the reaction rate [v1], maximum reaction
rate (Vmax), substrate concentration [S] and the Michaelis-Menton constant (Km).
The Michaelis-Menten equation can be used to
demonstrate that at the substrate concentration that produces exactly half of
the maximum reaction rate, i.e.,1/2 Vmax, the substrate concentration is
numerically equal to Km. This fact provides a simple yet powerful bioanalytical
tool that has been used to characterize both normal and altered enzymes, such
as those that produce the symptoms of genetic diseases. Rearranging the
Michaelis-Menton equation leads to:
From
this equation it should be apparent that when the substrate concentration is
half that required to support the maximum rate of reaction, the observed rate,
v1, will, be equal to Vmax divided by 2; in other words, v1 = [Vmax/2]. At this
substrate concentration Vmax/v1 will be exactly equal to 2, with the result
that:
[S](1)
= Km
The
latter is an algebraic statement of the fact that, for enzymes of the Michaelis-Menten
type, when the observed reaction rate is half of the maximum possible reaction
rate, the substrate concentration is numerically equal to the Michaelis-Menten
constant. In this derivation, the units of Km are those used to specify the
concentration of S, usually Molarity.
The
Michaelis-Menten equation has the same form as the equation for a rectangular
hyperbola; graphical analysis of reaction rate (v) versus substrate
concentration [S] produces a hyperbolic rate plot.
Plot of substrate concentration versus
reaction velocity
The key
features of the plot are marked by points A, B and C. At high substrate
concentrations the rate represented by point C the rate of the reaction is almost
equal to Vmax, and the difference in rate at nearby concentrations of substrate
is almost negligible. If the Michaelis-Menten plot is extrapolated to
infinitely high substrate concentrations, the extrapolated rate is equal to
Vmax. When the reaction rate becomes independent of substrate concentration, or
nearly so, the rate is said to be zero order. (Note that the reaction is zero
order only with respect to this substrate. If the reaction has two substrates,
it may or may not be zero order with respect to the second substrate). The very
small differences in reaction velocity at substrate concentrations around point
C (near Vmax) reflect the fact that at these concentrations almost all of the
enzyme molecules are bound to substrate and the rate is virtually independent
of substrate, hence zero order. At lower substrate concentrations, such
as at points A and B, the lower reaction velocities indicate that at any moment
only a portion of the enzyme molecules are bound to the substrate. In fact, at
the substrate concentration denoted by point B, exactly half the enzyme
molecules are in an ES complex at any instant and the rate is exactly one half
of Vmax. At substrate concentrations near point A the rate appears to be
directly proportional to substrate concentration, and the reaction rate is said
to be first order.
Inhibition of Enzyme Catalyzed
Reactions
To
avoid dealing with curvilinear plots of enzyme catalyzed reactions, biochemists
Lineweaver and Burk introduced an analysis of enzyme kinetics based on the
following rearrangement of the Michaelis-Menten equation:
[1/v]
= [Km (1)/ Vmax[S] + (1)/Vmax]
Plots
of 1/v versus 1/[S] yield straight lines having a slope of Km/Vmax and an intercept
on the ordinate at 1/Vmax.
A Lineweaver-Burk Plot
An
alternative linear transformation of the Michaelis-Menten equation is the
Eadie-Hofstee transformation:
v/[S]
= -v [1/Km] + [Vmax/Km]
and
when v/[S] is plotted on the y-axis versus v on the x-axis, the result is a
linear plot with a slope of -1/Km and the value Vmax/Km as the intercept on the
y-axis and Vmax as the intercept on the x-axis.
Both the Lineweaver-Burk and Eadie-Hofstee
transformation of the Michaelis-Menton equation are useful in the analysis of
enzyme inhibition. Since most clinical drug therapy is based on inhibiting the
activity of enzymes, analysis of enzyme reactions using the tools described
above has been fundamental to the modern design of pharmaceuticals. Well- known
examples of such therapy include the use of methotrexate in cancer chemotherapy
to semi-selectively inhibit DNA synthesis of malignant cells, the use of
aspirin to inhibit the synthesis of prostaglandins which are at least partly
responsible for the aches and pains of arthritis, and the use of sulfa drugs to
inhibit the folic acid synthesis that is essential for the metabolism and
growth of disease-causing bacteria. In addition, many poisons,
such as cyanide, carbon monoxide and polychlorinated biphenols (PCBs). produce
their life- threatening effects by means of enzyme inhibition.
The major contribution of Michaelis and Menten was to think of enzyme
reactions in two stages. In the first, the substrate binds reversibly to the
enzyme, forming the enzyme-substrate complex. This is sometimes called the
Michaelis-Menten complex in their honor. The enzyme then catalyzes the chemical
step in the reaction and releases the product.
Saturation curve
for an enzyme reaction showing the relation between the substrate concentration
(S) and rate (v).Enzymes can catalyze up to several million reactions per
second. For example, the reaction catalyzed by orotidine 5'-phosphate
decarboxylase will consume half of its substrate in 78 million years if no
enzyme is present. However, when the decarboxylase is added, the same process
takes just 25 milliseconds. Enzyme rates depend on solution conditions and
substrate concentration. Conditions that denature the protein abolish enzyme
activity, such as high temperatures, extremes of pH or high salt
concentrations, while raising substrate concentration tends to increase
activity. To find the maximum speed of an enzymatic reaction, the substrate
concentration is increased until a constant rate of product formation is seen.
This is shown in the saturation curve, shown on the right. Saturation happens because, as substrate concentration increases, more
and more of the free enzyme is converted into the substrate-bound ES form. At
the maximum velocity (Vmax) of the enzyme, all enzyme active sites are
saturated with substrate, and the amount of ES complex is the same as the total
amount of enzyme.
However, Vmax is only one kinetic constant of enzymes. The amount of substrate
needed to achieve a given rate of reaction is also important. This is given by
the Michaelis-Menten constant (Km), which is the substrate concentration
required for an enzyme to reach one-half its maximum velocity. Each enzyme has
a characteristic Km for a given substrate, and this can show how tight the
binding of the substrate is to the enzyme. Another useful constant is kcat,
which is the number of substrate molecules handled by one active site per
second.
The defficiency of an enzyme can be expressed in terms of kcat/Km. This
is also called the specificity constant and incorporates the rate constants for
all steps in the reaction. Because the specificity constant reflects both
affinity and catalytic ability, it is useful for comparing different enzymes
against each other, or the same enzyme with different substrates. The
theoretical maximum for the specificity constant is called the diffusion limit
and is about 108 to 109 (M-1 s-1). At this point every collision of the enzyme
with its substrate will result in catalysis, and the rate of product formation
is not limited by the reaction rate but by the diffusion rate. Enzymes with
this property are called catalytically perfect or kinetically perfect. Example
of such enzymes are triose-phosphate isomerase, carbonic anhydrase,
acetylcholinesterase, catalase, fumarase, ß-lactamase, and superoxide
dismutase.
Some enzymes operate with kinetics which are faster than diffusion
rates, which would seem to be impossible. Several
mechanisms have been invoked to explain this phenomenon. Some proteins are
believed to accelerate catalysis by drawing their substrate in and
pre-orienting them by using dipolar electric fields. Other models invoke a
quantum-mechanical tunneling explanation, whereby a proton or an electron can
tunnel through activation barriers, although for proton tunneling this model
remains somewhat controversial. Quantum tunneling for protons has been observed
in tryptamine. This suggests that enzyme catalysis may be more
accurately characterized as "through the barrier" rather than the
traditional model, which requires substrates to go "over" a lowered
energy barrier.
Enzyme
inhibitors fall into two broad classes: those causing irreversible inactivation
of enzymes and those whose inhibitory effects can be reversed. Inhibitors of
the first class usually cause an inactivating, covalent modification of enzyme
structure. Cyanide is a classic example of an irreversible enzyme inhibitor: by
covalently binding mitochondrial cytochrome oxidase, it inhibits all the
reactions associated with electron transport. The kinetic effect of
irreversible inhibitors is to decrease the concentration of active enzyme, thus
decreasing the maximum possible concentration of ES complex. Since the limiting
enzyme reaction rate is often k2[ES], it is clear that under these
circumstances the reduction of enzyme concentration will lead to decreased
reaction rates. Note that when enzymes in cells are only partially inhibited by
irreversible inhibitors, the remaining unmodified enzyme molecules are not
distinguishable from those in untreated cells; in particular, they have the
same turnover number and the same Km. Turnover number, related to Vmax, is
defined as the maximum number of moles of substrate that can be converted to
product per mole of catalytic site per second. Irreversible inhibitors are
usually considered to be poisons and are generally unsuitable for therapeutic
purposes.
Reversible inhibitors can be divided into two main categories; with a third category, uncompetitive
inhibitors, rarely encountered.
Inhibitor Type |
Binding Site on Enzyme |
Kinetic effect |
Competitive Inhibitor |
Specifically at the catalytic site,
where it competes with substrate for binding in a dynamic equilibrium- like
process. Inhibition is
reversible by substrate. |
Vmax is unchanged; Km,
as defined by [S] required for 1/2 maximal activity, is increased. |
Noncompetitive Inhibitor |
Binds E or ES complex other than at
the catalytic site. Substrate binding unaltered, but ESI complex cannot form
products. Inhibition
cannot be reversed by substrate. |
Km appears unaltered; Vmax
is decreased proportionately to inhibitor concentration. |
Uncompetitive Inhibitor |
Binds only to ES complexes at locations
other than the catalytic site. Substrate binding modifies enzyme structure,
making inhibitor- binding site available. Inhibition cannot be reversed by substrate. |
Apparent Vmax decreased;
Km, as defined by [S] required for 1/2 maximal activity, is
decreased. |
Inhibitor
Type
Specifically
at the catalytic site, where it competes with substrate for binding in a
dynamic equilibrium- like process. Inhibition is reversible by substrate.
Vmax is unchanged; Km, as defined by [S] required
for 1/2 maximal activity, is increased.
http://www.youtube.com/watch?v=0pJOFze055Y
Noncompetitive
Inhibitor
Binds E or ES complex other than at the
catalytic site. Substrate binding unaltered, but ESI complex cannot form
products. Inhibition cannot be reversed by substrate.
Km appears unaltered; Vmax is decreased
proportionately to inhibitor concentration.
Uncompetitive Inhibitor
Binds only to ES complexes at locations other
than the catalytic site. Substrate binding modifies enzyme structure, making
inhibitor- binding site available. Inhibition cannot be reversed by substrate.
Apparent Vmax decreased; Km, as defined by [S]
required for 1/2 maximal activity, is decreased.
The hallmark
of all the reversible inhibitors is that when the inhibitor concentration
drops, enzyme activity is regenerated. Usually these inhibitors bind to enzymes
by non-covalent forces and the inhibitor maintains a reversible equilibrium
with the enzyme. The equilibrium constant for the dissociation of enzyme
inhibitor complexes is known as KI:
KI
= [E][I]/[E--I--complex]
The
importance of KI is that in all enzyme reactions where substrate, inhibitor and
enzyme interact, the normal Km and or Vmax for substrate enzyme interaction
appear to be altered. These changes are a consequence of the influence of KI on
the overall rate equation for the reaction. The effects of KI are best observed
in Lineweaver-Burk plots.
Probably the best known reversible inhibitors are
competitive inhibitors, which always bind at the catalytic or active site of
the enzyme. Most drugs that alter enzyme activity are of this type. Competitive
inhibitors are especially attractive as clinical modulators of enzyme activity
because they offer two routes for the reversal of enzyme inhibition, while
other reversible inhibitors offer only one. First, as with all kinds of
reversible inhibitors, a decreasing concentration of the inhibitor reverses the
equilibrium regenerating active free enzyme. Second, since substrate and
competitive inhibitors both bind at the same site they compete with one another
for binding .
Raising
the concentration of substrate (S), while holding the concentration of
inhibitor constant, provides the second route for reversal of competitive
inhibition. The greater the proportion of substrate, the greater the proportion
of enzyme present in competent ES complexes.
As
noted earlier, high concentrations of substrate can displace virtually all
competitive inhibitor bound to active sites. Thus, it is apparent that Vmax
should be unchanged by competitive inhibitors. This characteristic of
competitive inhibitors is reflected in the identical vertical-axis intercepts
of Lineweaver-Burk plots, with and without inhibitor.
Since
attaining Vmax requires appreciably higher substrate concentrations in the
presence of competitive inhibitor, Km (the substrate concentration at half
maximal velocity) is also higher, as demonstrated by the differing negative
intercepts on the horizontal axis in panel B.
Analogously, panel C illustrates that noncompetitive
inhibitors appear to have no effect on the intercept at the x-axis implying
that noncompetitive inhibitors have no effect on the Km of the enzymes they
inhibit. Since noncompetitive inhibitors do not interfere in the equilibration
of enzyme, substrate and ES complexes, the Km's of Michaelis-Menten type
enzymes are not expected to be affected by noncompetitive inhibitors, as
demonstrated by x-axis intercepts in panel C. However, because complexes that
contain inhibitor (ESI) are incapable of progressing to reaction products, the
effect of a noncompetitive inhibitor is to reduce the concentration of ES
complexes that can advance to product. Since Vmax = k2[Etotal], and
the concentration of competent Etotal is diminished by the amount of ESI
formed, noncompetitive inhibitors are expected to decrease Vmax, as illustrated
by the y-axis intercepts in panel C.
A
corresponding analysis of uncompetitive inhibition leads to the expectation that
these inhibitors should change the apparent values of Km as well as Vmax.
Changing both constants leads to double reciprocal plots, in which intercepts
on the x and y axes are proportionately changed; this leads to the production
of parallel lines in inhibited and uninhibited reactions.
Inhibition
Competitive inhibitors bind reversibly to the enzyme, preventing the
binding of substrate. On the other hand, binding of substrate prevents binding
of the inhibitor. Substrate and inhibitor compete for the enzyme.Main article:
Enzyme inhibitor
Enzyme reaction rates can be decreased by various types of enzyme
inhibitors.
Reversible inhibitors
In competitive inhibition the inhibitor binds to the substrate binding
site (figure right, top, thus preventing substrate from binding (EI complex).
Often competitive inhibitors strongly resemble the real substrate of the
enzyme. For example, methotrexate is a competitive inhibitor of the enzyme
dihydrofolate reductase, which catalyzes the reduction of dihydrofolate to tetrahydrofolate.
The similarity between the structures of folic acid and this drug are shown in
the figure to the right bottom.
Non-competitive inhibition
In order to do its work, an enzyme must unite - even if ever so briefly - with
at least one of the reactants. In most cases, the forces that hold the enzyme
and its substrate are noncovalent, an assortment of:
·
and hydrophobic interactions
Link
to discussion of the noncovalent forces that hold macromolecules |
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.
This requirement for complementarity in the configuration of substrate and
enzyme explains the remarkable specificity of most enzymes. Generally, a
given enzyme is able to catalyze only a single chemical reaction or, at most, a
few reactions involving substrates sharing the same general structure.
The necessity for a
close, if brief, fit between enzyme and substrate explains the phenomenon of
competitive inhibition.
It catalyzes the oxidation (by the removal of
two hydrogen atoms) of succinic acid (a). If one adds malonic acid
to cells, or to a test tube mixture of succinic acid and the enzyme, the action
of the enzyme is strongly inhibited. This is because the structure of malonic
acid allows it to bind to the same site on the enzyme (b). But there is
no oxidation so no speedy release of products.
The inhibition is called competitive because if you increase the ratio of
succinic to malonic acid in the mixture, you will gradually restore the rate of
catalysis. At a 50:1 ratio, the two molecules compete on roughly equal terms
for the binding (=catalytic) site on the enzyme.
Non-competitive inhibitors can bind either to the active site, or to
other parts of the enzyme far away from the substrate-binding site. Moreover,
non-competitive inhibitors bind to the enzyme-substrate (ES) complex and to the
free enzyme. Their binding to this site changes the shape of the enzyme and
stops the active site binding substrate(s). Consequently, since there is no
direct competition between the substrate and inhibitor for the enzyme, the
extent of inhibition depends only on the inhibitor concentration and will not
be affected by the substrate concentration.
Biological function
Enzymes serve a wide variety of functions inside living organisms. They
are indispensable for signal transduction and cell regulation, often via
kinases and phosphatases. They also generate movement, with myosin hydrolysing
ATP to generate muscle contraction and also moving cargo around the cell as
part of the cytoskeleton. Other ATPases in the cell membrane are ion pumps
involved in active transport. Enzymes are also involved in more exotic
functions, such as luciferase generating light in fireflies.
Viruses can contain enzymes for infecting cells, such as the HIV
integrase and reverse transcriptase, or for viral release from cells, like the
influenza virus neuraminidase.
An important function of enzymes is in the digestive systems of animals.
Enzymes such as amylases and proteases break down large molecules (starch or
proteins, respectively) into smaller ones, so they can be absorbed by the
intestines. Starch is inabsorbable in the intestine but enzymes hydrolyse the
starch chains into smaller molecules such as maltose and eventually glucose,
which can then be absorbed. Different enzymes digest different food substances.
In ruminants which have a herbivorous diets, bacteria in the gut produce
another enzyme, cellulase to break down the cellulose cell walls of plant
fiber.
Several enzymes can work together in a specific order, creating
metabolic pathways. In a metabolic pathway, one enzyme takes the product of
another enzyme as a substrate. After the catalytic reaction, the product is
then passed on to another enzyme. Sometimes more than one enzyme can catalyse
the same reaction in parallel, this can allow more complex regulation: with for
example a low constant activity being provided by one enzyme but an inducible
high activity from a second enzyme.
Enzymes determine what steps occur in these pathways. Without enzymes,
metabolism would neither progress through the same steps, nor be fast enough to
serve the needs of the cell. Indeed, a metabolic pathway such as glycolysis
could not exist independently of enzymes. Glucose, for example, can react
directly with ATP to become phosphorylated at one or more of its carbons. In
the absence of enzymes, this occurs so slowly as to be insignificant. However,
if hexokinase is added, these slow reactions continue to take place except that
phosphorylation at carbon 6 occurs so rapidly that if the mixture is tested a
short time later, glucose-6-phosphate is found to be the only significant product.
Consequently, the network of metabolic pathways within each cell depends on the
set of functional enzymes that are present.
Control activity
There are five main ways that enzyme activity is controlled in the cell.
Regulation
of Enzyme Activity
While
it is clear that enzymes are responsible for the catalysis of almost all
biochemical reactions, it is important to also recognize that rarely, if ever,
do enzymatic reactions proceed in isolation. The most common scenario is that
enzymes catalyze individual steps of multi-step metabolic pathways, as is the
case with glycolysis, gluconeogenesis or the synthesis of fatty acids. As a
consequence of these lock- step sequences of reactions, any given enzyme is
dependent on the activity of preceding reaction steps for its substrate.
In
humans, substrate concentration is dependent on food supply and is not usually
a physiologically important mechanism for the routine regulation of enzyme
activity. Enzyme concentration, by contrast, is continually modulated in response
to physiological needs. Three principal mechanisms are known to regulate the
concentration of active enzyme in tissues:
1.
Regulation of gene expression controls the quantity and rate of enzyme
synthesis.
2. Proteolytic enzyme activity determines the
rate of enzyme degradation.
3.
Covalent modification of preexisting pools of inactive proenzymes produces
active enzymes.
Enzyme
synthesis and proteolytic degradation are comparatively slow mechanisms for
regulating enzyme concentration, with response times of hours, days or even
weeks. Proenzyme activation is a more rapid method of increasing enzyme
activity but, as a regulatory mechanism, it has the disadvantage of not being a
reversible process. Proenzymes are generally synthesized in abundance, stored
in secretory granules and covalently activated upon release from their storage
sites. Examples of important proenzymes include pepsinogen, trypsinogen and
chymotrypsinogen, which give rise to the proteolytic digestive enzymes.
Likewise, many of the proteins involved in the cascade of chemical reactions
responsible for blood clotting are synthesized as proenzymes. Other important
proteins, such as peptide hormones and collagen, are also derived by covalent
modification of precursors.
Another
mechanism of regulating enzyme activity is to sequester enzymes in compartments
where access to their substrates is limited. For example, the proteolysis of
cell proteins and glycolipids by enzymes responsible for their degradation is
controlled by sequestering these enzymes within the lysosome.
In
contrast to regulatory mechanisms that alter enzyme concentration, there is an
important group of regulatory mechanisms that do not affect enzyme
concentration, are reversible and rapid in action, and actually carry out most
of the moment- to- moment physiological regulation of enzyme activity. These
mechanisms include allosteric regulation, regulation by reversible covalent
modification and regulation by control proteins such as calmodulin. Reversible
covalent modification is a major mechanism for the rapid and transient
regulation of enzyme activity. The best examples, again, come from studies on
the regulation of glycogen metabolism where phosphorylation of glycogen
synthase and glycogen phosphorylase kinase results in the stimulation of
glycogen degradation while glycogen synthesis is coordinately inhibited.
Numerous other enzymes of intermediary metabolism are affected by
phosphorylation, either positively or negatively. These covalent
phosphorylations can be reversed by a separate sub-subclass of enzymes known as
phosphatases. Recent research has indicated that the aberrant phosphorylation
of growth factor and hormone receptors, as well as of proteins that regulate
cell division, often leads to unregulated cell growth or cancer. The usual
sites for phosphate addition to proteins are the serine, threonine and tyrosine
R group hydroxyl residues.
Enzyme production (transcription and
translation of enzyme genes) can be enhanced or diminished by a cell in
response to changes in the cell's environment. This form of gene regulation is
called enzyme induction and inhibition. For example, bacteria may become
resistant to antibiotics such as penicillin because enzymes called
beta-lactamases are induced that hydrolyse the crucial beta-lactam ring within
the penicillin molecule. Another example are enzymes in the liver called
cytochrome P450 oxidases, which are important in drug metabolism. Induction or
inhibition of these enzymes can cause drug interactions.
Enzymes can be compartmentalized, with
different metabolic pathways occurring in different cellular compartments. For
example, fatty acids are synthesized by one set of enzymes in the cytosol,
endoplasmic reticulum and the Golgi apparatus and used by a different set of enzymes
as a source of energy in the mitochondrion, through β-oxidation.
Enzymes can be regulated by inhibitors and
activators. For example, the end product(s) of a
metabolic pathway are often inhibitors for one of the first enzymes of the
pathway (usually the first irreversible step, called committed step), thus
regulating the amount of end product made by the pathways. Such a regulatory
mechanism is called a negative feedback mechanism, because the amount of the
end product produced is regulated by its own concentration. Negative feedback
mechanism can effectively adjust the rate of synthesis of intermediate
metabolites according to the demands of the cells. This helps allocate
materials and energy economically, and prevents the manufacture of excess end products.
Like other homeostatic devices, the control of enzymatic action helps to
maintain a stable internal environment in living organisms.
Enzymes can be regulated through post-translational modification. This can
include phosphorylation, myristoylation and glycosylation. For example, in the
response to insulin, the phosphorylation of multiple enzymes, including
glycogen synthase, helps control the synthesis or degradation of glycogen and
allows the cell to respond to changes in blood sugar. Another example of
post-translational modification is the cleavage of the polypeptide chain.
Chymotrypsin, a digestive protease, is produced in inactive form as
chymotrypsinogen in the pancreas and transported in this form to the stomach
where it is activated. This stops the enzyme from digesting the pancreas or
other tissues before it enters the gut. This type of inactive precursor to an
enzyme is known as a zymogen.
Some enzymes may become activated when localized to a different
environment (eg. from a reducing (cytoplasm) to an oxidising (periplasm)
environment, high pH to low pH etc). For example, hemagglutinin of the
influenza virus undergoes a conformational change once it encounters the acidic
environment of the host cell vesicle causing its activation.
Involvement in disease Phenylalanine
hydroxylase. Created from PDB 1KW0Since the tight control of enzyme activity is
essential for homeostasis, any malfunction (mutation, overproduction, underproduction
or deletion) of a single critical enzyme can lead to a genetic disease. The
importance of enzymes is shown by the fact that a lethal illness can be caused
by the malfunction of just one type of enzyme out of the thousands of types
present in our bodies.
One example is the most common type of phenylketonuria. A mutation of a
single amino acid in the enzyme phenylalanine hydroxylase, which catalyzes the
first step in the degradation of phenylalanine, results in build-up of
phenylalanine and related products. This can lead to mental retardation if the
disease is untreated.
Another example is when germline mutations in genes coding for DNA
repair enzymes cause hereditary cancer syndromes such as xeroderma pigmentosum.
Defects in these enzymes cause cancer since the body is less able to repair
mutations in the genome. This causes a slow accumulation of mutations and
results in the development of many types of cancer in the sufferer.
Naming conventions
An enzyme's name is often derived from its substrate or the chemical
reaction it catalyzes, with the word ending in -ase. Examples are lactase,
alcohol dehydrogenase and DNA polymerase. This may result in different enzymes,
called isoenzymes, with the same function having the same basic name. Isoenzymes
have a different amino acid sequence and might be distinguished by their
optimal pH, kinetic properties or immunologically. Furthermore, the normal
physiological reaction an enzyme catalyzes may not be the same as under
artifical conditions. This can result in the same enzyme being identified with
two different names. E.g. Glucose isomerase, used industrially to convert
glucose into the sweetener fructose, is a xylose isomerase in vivo.
Allosteric Enzymes
Allosteric modulation
Allosteric enzymes change their structure in response to binding of
effectors. Modulation can be direct, where the effector binds directly to
binding sites in the enzyme, or indirect, where the effector binds to other
proteins or protein subunits that interact with the allosteric enzyme and thus
influence catalytic activity.
In
addition to simple enzymes that interact only with substrates and inhibitors,
there is a class of enzymes that bind small, physiologically important
molecules and modulate activity in ways other than those described above. These
are known as allosteric enzymes; the small regulatory molecules to which they
bind are known as effectors. Allosteric effectors bring about catalytic
modification by binding to the enzyme at distinct allosteric sites, well
removed from the catalytic site, and causing conformational changes that are
transmitted through the bulk of the protein to the catalytically active
site(s).
The
hallmark of effectors is that when they bind to enzymes, they alter the
catalytic properties of an enzyme's active site. Those that increase catalytic
activity are known as positive effectors. Effectors that reduce or inhibit
catalytic activity are negative effectors.
Most
allosteric enzymes are oligomeric (consisting of multiple subunits); generally
they are located at or near branch points in metabolic pathways, where they are
influential in directing substrates along one or another of the available
metabolic paths. The effectors that modulate the activity of these allosteric
enzymes are of two types. Those activating and inhibiting effectors that bind
at allosteric sites are called heterotropic effectors. (Thus there exist both
positive and negative heterotropic effectors.) These effectors can assume a
vast diversity of chemical forms, ranging from simple inorganic molecules to
complex nucleotides such as cyclic adenosine monophosphate (cAMP). Their single
defining feature is that they are not identical to the substrate.
In
many cases the substrate itself induces distant allosteric effects when it
binds to the catalytic site. Substrates acting as effectors are said to be
homotropic effectors. When the substrate is the effector, it can act as such,
either by binding to the substrate-binding site, or to an allosteric effector
site. When the substrate binds to the catalytic site it transmits an
activity-modulating effect to other subunits of the molecule. Often used as the
model of a homotropic effector is hemoglobin, although it is not a branch-point
enzyme and thus does not fit the definition on all counts.
There are two ways that enzymatic activity can be
altered by effectors: the Vmax can be increased or decreased, or the Km can be
raised or lowered. Enzymes whose Km is altered by effectors are said to be
K-type enzymes and the effector a K-type effector. If Vmax is altered, the
enzyme and effector are said to be V-type. Many allosteric enzymes respond to
multiple effectors with V-type and K-type behavior. Here again, hemoglobin is
often used as a model to study allosteric interactions, although it is not strictly
an enzyme.
In the preceding discussion we assumed that allosteric
sites and catalytic sites were homogeneously present on every subunit of an
allosteric enzyme. While this is often the case, there is another class of
allosteric enzymes that are comprised of separate catalytic and regulatory
subunits. The archetype of this class of enzymes is
cAMP-dependent protein kinase (PKA), whose mechanism of activation is
illustrated in the Figure below. The enzyme is tetrameric, containing two
catalytic subunits and two regulatory subunits, and enzymatically inactive.
When intracellular cAMP levels rise, one molecule of cAMP binds to each
regulatory subunit, causing the tetramer to dissociate into one regulatory
dimer and two catalytic monomers. In the dissociated form, the catalytic
subunits are fully active; they catalyze the phosphorylation of a number of
other enzymes, such as those involved in regulating glycogen metabolism. The
regulatory subunits have no catalytic activity.
Representative
pathway for the activation of cAMP-dependent protein kinase (PKA). In this
example glucagon binds to its' cell-surface receptor, thereby activating the
receptor. Activation of the receptor is coupled to the activation of a
receptor-coupled G-protein (GTP-binding and hydrolyzing protein) composed of 3
subunits. Upon activation the a-subunit dissociates and binds to and activates
adenylate cyclase. Adenylate cylcase then converts ATP to cyclic-AMP (cAMP).
The cAMP thus produced then binds to the regulatory subunits of PKA leading to
dissociation of the associated catalytic subunits. The catalytic subunits are
inactive until dissociated from the regulatory subunits. Once released the
catalytic subunits of PKA phosphorylate numerous substrate using ATP as the
phosphate donor.
The 19th-century physiologist
Claude Bernard enunciated the conceptual
basis for metabolic regulation. He observed that
living organisms respond in ways that are both
quantitatively and temporally appropriate to permit them to survive
the multiple challenges posed by changes in their
external and internal environments. Walter Cannon
subsequently coined the term “homeostasis” to
describe the ability of animals to maintain a constant
intracellular environment despite changes in their
external environment. We now know that organisms respond
to changes in their external and internal environment by
balanced, coordinated changes in the rates of
specific metabolic reactions. Many human diseases, including
cancer, diabetes, cystic fibrosis, and Alzheimer’s
disease, are characterized by regulatory dysfunctions triggered
by pathogenic agents or genetic mutations. For
example, many oncogenic viruses elaborate protein-tyrosine
kinases that modify the regulatory events which
control patterns of gene expression, contributing to the
initiation and progression of cancer. The toxin from Vibrio
cholerae, the causative agent of cholera, disables
sensor-response pathways in intestinal epithelial cells by
ADP-ribosylating the GTP-binding proteins (G-proteins)
that link cell surface receptors to adenylyl cyclase.
The consequent activation of the cyclase triggers the flow
of water into the intestines, resulting in massive diarrhea
and dehydration. Yersinia pestis, the causative agent of
plague, elaborates a protein-tyrosine phosphatase that
hydrolyzes phosphoryl groups on key cytoskeletal proteins.
Knowledge of factors that control the rates of
enzyme-catalyzed reactions thus is essential to an
understanding
of the molecular basis of disease.
Factors Affecting Enzyme Action
The
activity of enzymes is strongly affected by changes in pH and temperature.
Each enzyme works best at a certain pH (left graph) and temperature (right
graph), its activity decreasing at values above and below that point. This is
not surprising considering the importance of
·
tertiary structure (i.e.
shape) in enzyme function and noncovalent
forces, e.g., ionic interactions and hydrogen bonds, in determining that shape.
Examples:
·
the protease pepsin works best as a pH of 1-2
(found in the stomach) while
·
the protease trypsin is inactive at such a low pH
but very active at a pH of 8 (found in the small intestine as the bicarbonate
of the pancreatic fluid neutralizes the arriving stomach contents).
Changes in pH alter
the state of ionization of charged amino acids (e.g., Asp, Lys) that may play a
crucial role in substrate binding and/or the catalytic action itself. Without
the unionized -COOH group of Glu-35
and the ionized -COO- of Asp-52,
the catalytic action of lysozyme would cease.
Hydrogen bonds are
easily disrupted by increasing temperature. This, in turn, may disrupt the
shape of the enzyme so that its affinity for its substrate diminishes. The
ascending portion of the temperature curve (red arrow in right-hand graph
above) reflects the general effect of
increasing temperature on the rate of chemical reactions (graph at
left). The descending portion of the curve above (blue arrow) reflects the loss
of catalytic activity as the enzyme molecules become denatured at high
temperatures.
The accumulation
of a substance within a cell may specifically activate (blue arrow) an enzyme
that sets in motion a sequence of reactions for which that substance is the
initial substrate. This reduces the concentration of the initial substrate.
In the case if
feedback inhibition and precursor activation, the activity of the enzyme is
being regulated by a molecule which is not
its substrate. In these cases, the regulator molecule binds to the enzyme at a
different site than the one to which the substrate binds. When the regulator
binds to its site, it alters the shape of the enzyme so that its activity is
changed. This is called an allosteric
effect.
·
In feedback inhibition, the
allosteric effect lowers the affinity of the enzyme for its substrate.
·
In precursor activation, the
regulator molecule increases the affinity of the enzyme in the series for its
substrate.
Regulation of Enzyme Synthesis
The four mechanisms described above
regulate the activity of enzymes already present within the cell.
What about enzymes that are not needed
or are needed but not present?
Here, too, control mechanisms are at
work that regulate the rate at which new enzymes are synthesized. Most of these
controls work by turning on - or off - the transcription of genes.
If, for example, ample quantities of an
amino acid are already available to the cell from its extracellular fluid,
synthesis of the enzymes that would enable the cell to produce that amino acid
for itself is shut down.
Conversely, if a new
substrate is made available to the cell, it may induce the synthesis of the
enzymes needed to cope with it. Yeast cells, for example, do not ordinarily
metabolize lactose and no lactase
can be detected in them. However, if grown in a medium containing lactose, they
soon begin synthesizing lactase - by transcribing and translating the necessary
gene(s) - and so can begin to metabolize the sugar.
E. coli also has a mechanism which
regulates enzyme synthesis by controlling translation of a needed messenger RNA.
The Role of
Ezymes in Biological Reactions
The laws of
thermodymatics apply to chemical reactions anywhere in the universe. No
reactions can violate the rules of thermodymatics. All reaction must proceed to
a level of minimum energy and maximum entropy or have a favorable balance
between the two. Enzyme simply increase the rate (rate is equal to the number
of reactant molecules converted to product per unit of time) at which
spontaneous reactions take place. Enzymes cannot make a reaction occur that
would not proceed spontaneously without the enzyme. The same principle apply to
reversible reaction. Enzymes do not alter the equilibrium point of reversible
reaction.
Most biological
reactions would take place to slow without enzyme for life to exist. The rates
would be essentially zero at biological temperatures. For example the oxidation
of glucose to CO2 and H2O is spontaneous and proceeds
almost completely in the direction stated. However, without enzymes, glucose
oxidation occurs do slowly at physiological temperatures that the rate is
essentially unmeasureable.
The increases in rate achieve by
enzymes, depending on the enzyme and reaction, range from a minimum of about a
million to as much as a trillion times faster than the uncatalyzed reactions at
equivalent concentrations and temperatures.
Enzyme and
Activation Energy
Activation energy is
the energy barrier over which the molecules in a system must be raised for a reaction
to take place (Figure 1).
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.
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.
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.
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.
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).
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.
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.
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.
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
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.
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.
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).
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.
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.
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.
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.
Top:
general diagram showing competitor in the active site normally occupied by the
natural substrate; Bottom: specific case of succinate dehydrogenase and its
natural substrate (succinate) and competitors (oxalate et al.).
Noncompetitive
Inhibition
occurs when the inhibitory chemical, which does not have to resemble the
substrate, binds to the enzyme other than at the active site. Lead binds to SH
groups in this fashion. Irreversible Inhibition occurs when the chemical either
permanently binds to or massively denatures the enzyme so that the tertiary
structure cannot be restored. Nerve gas permanently blocks pathways involved in
nerve message transmission, resulting in death. Penicillin, the first of the "wonder
drug" antibiotics, permanently blocks the pathways certain bacteria use to
assemble their cell wall
components.
The four
mechanisms described above regulate the activity of enzymes already present
within the cell.
What about enzymes
that are not needed or are needed but not present?
Here, too,
control mechanisms are at work that regulate the rate at which new enzymes are
synthesized. Most of these controls work by turning on — or off — the transcription of genes.
If, for example, ample
quantities of an amino acid are already available to the cell from its
extracellular fluid, synthesis of the enzymes that would enable the cell to
produce that amino acid for itself is shut down.
Conversely,
if a new substrate is made available to the cell, it may induce the synthesis
of the enzymes needed to cope with it. Yeast cells, for example, do not ordinarily
metabolize lactose and no lactase can be detected in them. However, if grown in
a medium containing lactose, they soon begin synthesizing lactase — by
transcribing and translating the necessary gene(s) — and so can begin to
metabolize the sugar.
REGULATION
OF METABOLITE FLOW CAN BE ACTIVE OR PASSIVE
Enzymes that operate at their maximal rate cannot respond to an
increase in substrate concentration, and can respond only
to a precipitous decrease in substrate concentration. For
most enzymes, therefore, the average intracellular
concentration of their substrate tends to be close to
the Km value, so that changes in substrate concentration
generate corresponding changes in metabolite
flux. Responses to changes in substrate level
represent an important but passive means for coordinating
metabolite flow and maintaining homeostasis in
quiescent cells. However, they offer limited scope
for responding to changes in environmental variables. The mechanisms
that regulate enzyme activity in an active manner
in response to internal and external signals are
discussed below.
Metabolite Flow
Tends to Be Unidirectional
Despite the existence of short-term oscillations in metabolite
concentrations and enzyme levels, living cells exist in a
dynamic steady state in which the mean
concentrations of metabolic intermediates remain relatively constant
over time. While all chemical reactions are to
some extent reversible, in living cells the reaction
products serve as substrates for—and are removed by—other
enzyme-catalyzed reactions.
Many nominally reversible reactions thus occur unidirectionally. This succession of coupled metabolic
reactions is accompanied by an overall change in free energy that favors unidirectional metabolite flow. The unidirectional flow of metabolites
through a pathway with a large overall negative change in free energy is analogous to the flow of water through
a pipe in which one end is lower than the other. Bends or kinks in the pipe simulate individual
enzyme-catalyzed steps with a small negative or positive change in free energy. Flow of water through the pipe nevertheless
remains unidirectional due to the overall change in height, which corresponds to the overall change in free energy
in a pathway.
COMPARTMENTATION
ENSURES METABOLIC
EFFICIENCY
&
SIMPLIFIES REGULATION
In eukaryotes, anabolic and catabolic pathways that interconvert common
products may take place in specific subcellular
compartments. For example, many of the enzymes that
degrade proteins and polysaccharides reside inside
organelles called lysosomes. Similarly, fatty acid
biosynthesis occurs in the cytosol, whereas fatty acid
oxidation takes place within mitochondria.
Segregation of certain metabolic pathways within
specialized cell types can provide further physical
compartmentation. Alternatively, possession of
one or more unique intermediates can permit apparently opposing
pathways to coexist even in the absence of physical
barriers. For example, despite many shared intermediates and
enzymes, both glycolysis and gluconeogenesis
are favored energetically. This cannot be true if all the
reactions were the same. If one pathway was favored energetically,
the other would be accompanied by
a change in free energy G equal in magnitude but opposite in sign.
Simultaneous spontaneity of both pathways results from
substitution of one or more reactions
by different reactions favored thermodynamically in the opposite
direction. The glycolytic enzyme phosphofructokinase is
replaced by the gluconeogenic enzyme
fructose-1,6-bisphosphatase. The ability of
enzymes to discriminate between the structurally
similar coenzymes NAD+ and NADP+ also results in a
form of compartmentation, since it segregates the
electrons of NADH that are destined for ATP generation
from those of NADPH that participate in the reductive
steps in many biosynthetic pathways.
Controlling
an Enzyme That Catalyzes a Rate-Limiting Reaction Regulates
an
Entire Metabolic Pathway
While the flux of metabolites through metabolic pathways involves
catalysis by numerous enzymes, active control of
homeostasis is achieved by regulation of only a small
number of enzymes. The ideal enzyme for regulatory intervention
is one whose quantity or catalytic efficiency dictates
that the reaction it catalyzes is slow relative to all
others in the pathway. Decreasing the catalytic
efficiency or the quantity of the catalyst for the “bottleneck”
or rate-limiting reaction immediately reduces metabolite
flux through the entire pathway. Conversely, an
increase in either its quantity or catalytic efficiency enhances
flux through the pathway as a whole.
For example, acetyl-CoA carboxylase catalyzes the synthesis of
malonyl-CoA, the first committed reaction of fatty
acid biosynthesis. When synthesis of
malonyl-CoA is inhibited, subsequent reactions of fatty acid
synthesis cease due to lack of substrates. Enzymes that
catalyze rate-limiting steps serve as natural “governors” of
metabolic flux. Thus, they constitute efficient targets
for regulatory intervention by drugs. For example, inhibition
by “statin” drugs of HMG-CoA reductase, which catalyzes
the rate-limiting reaction of cholesterogenesis, curtails
synthesis of cholesterol.
REGULATION
OF ENZYME QUANTITY
The catalytic capacity of the rate-limiting reaction in a metabolic
pathway is the product of the concentration of
enzyme molecules and their intrinsic catalytic efficiency. It
therefore follows that catalytic capacity can be influenced
both by changing the quantity of enzyme present and by
altering its intrinsic catalytic efficiency.
Control of Enzyme Synthesis
Enzymes whose concentrations remain essentially constant over
time are termed constitutive enzymes. By contrast,
the concentrations of many other enzymes depend upon the
presence of inducers, typically substrates or
structurally related compounds, that initiate their
synthesis. Escherichia coli grown on glucose will, for
example, only catabolize lactose after addition of a β-galactoside,
an inducer that initiates synthesis of a β-galactosidase
and a galactoside permease. Inducible
enzymes of humans include tryptophan pyrrolase, threonine
dehydrase, tyrosine-α-ketoglutarate aminotransferase,
enzymes of the urea cycle, HMG-CoA reductase, and
cytochrome P450. Conversely, an excess of a metabolite
may curtail synthesis of its cognate
enzyme via repression. Both induction and repression involve
cis elements, specific DNA sequences located upstream of
regulated genes, and trans-acting regulatory proteins.
Control of Enzyme Degradation
The absolute quantity of an enzyme reflects the net balance between
enzyme synthesis and enzyme degradation, where ks
and kdeg represent the rate constants for the
overall processes of synthesis and degradation, respectively.
Changes in both the ks and kdeg of specific enzymes
occur in human subjects.
Protein turnover represents the net result of enzyme synthesis
and degradation. By measuring the rates of incorporation
of 15N-labeled amino acids into protein
and the rates of loss of 15N from protein, Schoenheimer deduced
that body proteins are in a state of “dynamic equilibrium”
in which they are continuously
synthesized and degraded. Mammalian proteins are degraded both by
ATP and ubiquitin-dependent pathways and by
ATP-independent pathways.
Susceptibility to proteolytic degradation can be influenced by the
presence of ligands such as substrates, coenzymes, or
metal ions that alter protein conformation. Intracellular
ligands thus can influence the rates at which specific
enzymes are degraded.
Enzyme levels in mammalian tissues respond to a wide
range of physiologic, hormonal, or dietary factors. For
example, glucocorticoids increase the concentration of
tyrosine aminotransferase by stimulating ks, and glucagon—despite
its antagonistic physiologic effects— increases ks
fourfold to fivefold. Regulation of liver arginase can
involve changes either in ks or in kdeg. After
a protein-rich meal, liver arginase levels rise and arginine synthesis
decreases. Arginase levels also rise in
starvation, but here arginase degradation decreases,
whereas ks remains unchanged. Similarly, injection of
glucocorticoids and ingestion of tryptophan both
elevate levels of tryptophan oxygenase. While the
hormone raises ks for oxygenase synthesis, tryptophan specifically
lowers kdeg by stabilizing the oxygenase against
proteolytic digestion.
MULTIPLE
OPTIONS ARE AVAILABLE FOR REGULATING CATALYTIC ACTIVITY
In humans, the induction of protein synthesis is a complex multistep
process that typically requires hours to produce
significant changes in overall enzyme level. By contrast,
changes in intrinsic catalytic efficiency effected by
binding of dissociable ligands (allosteric regulation) or by covalent
modification achieve regulation of enzymic
activity within seconds. Changes in protein level
serve long-term adaptive requirements, whereas changes
in catalytic efficiency are best suited for rapid and
transient alterations in metabolite flux.
ALLOSTERIC
EFFECTORS REGULATE CERTAIN ENZYMES
Feedback inhibition refers to inhibition of an enzyme in a
biosynthetic pathway by an end product of that pathway.
For example, for the biosynthesis of D from A catalyzed
by enzymes Enz1 through Enz3, high concentrations
of D inhibit conversion of A to B. Inhibition
results not from the “backing up” of intermediates but from
the ability of D to bind to and inhibit Enz1. Typically,
D binds at an allosteric site spatially distinct
from the catalytic site of the target
enzyme. Feedback inhibitors thus are allosteric effectors and
typically bear little or no structural similarity to the substrates
of the enzymes they inhibit. In this example, the
feedback inhibitor D acts as a negative allosteric effector
of Enz1.
IS
NOT SYNONYMOUS WITH FEEDBACK INHIBITION
In both mammalian and bacterial cells, end products “feed
back” and control their own synthesis, in many instances
by feedback inhibition of an early biosynthetic enzyme.
We must, however, distinguish between feedback
regulation, a phenomenologic term devoid of
mechanistic implications, and feedback inhibition, a
mechanism for regulation of enzyme activity. For example, while
dietary cholesterol decreases hepatic synthesis of
cholesterol, this feedback regulation does not involve
feedback inhibition. HMG-CoA reductase, the rate-limiting
enzyme of cholesterologenesis, is affected,
but cholesterol does not feedback-inhibit its activity.
Regulation in response to dietary cholesterol involves curtailment
by cholesterol or a cholesterol metabolite of the
expression of the gene that encodes HMG-CoA reductase (enzyme
repression).
MANY
HORMONES ACT THROUGH ALLOSTERIC SECOND MESSENGERS
Nerve impulses—and binding of hormones to cell surface receptors—elicit
changes in the rate of enzymecatalyzed reactions within
target cells by inducing the release or synthesis of
specialized allosteric effectors called second
messengers. The primary or “first” messenger is the
hormone molecule or nerve impulse. Second messengers include
3′,5′-cAMP, synthesized from ATP by
the enzyme adenylyl cyclase in response to the hormone epinephrine,
and Ca2+, which is stored inside the endoplasmic reticulum
of most cells. Membrane depolarization resulting from a
nerve impulse opens a membrane channel that
releases calcium ion into the cytoplasm, where it binds
to and activates enzymes involved in the regulation of
contraction and the mobilization of stored
glucose from glycogen. Glucose then supplies the increased energy
demands of muscle contraction. Other second
messengers include 3′,5′-cGMP and polyphosphoinositols, produced
by the hydrolysis of inositol phospholipids by
hormone-regulated phospholipases.
REGULATORY
COVALENT MODIFICATIONS
CAN BE REVERSIBLE
OR IRREVERSIBLE
In mammalian cells, the two most common forms of covalent
modification are partial proteolysis and phosphorylation.
Because cells lack the ability to reunite the two
portions of a protein produced by hydrolysis of a
peptide bond, proteolysis constitutes an irreversible modification.
By contrast, phosphorylation is a reversible
modification process. The phosphorylation of proteins
on seryl, threonyl, or tyrosyl residues, catalyzed by
protein kinases, is thermodynamically spontaneous. Equally
spontaneous is the hydrolytic removal of these phosphoryl
groups by enzymes called protein phosphatases.
PROTEASES
MAY BE SECRETED AS CATALYTICALLY INACTIVE PROENZYMES
Certain proteins are synthesized and secreted as inactive precursor
proteins known as proproteins. The proproteins of
enzymes are termed proenzymes or zymogens. Selective
proteolysis converts a proprotein by one or more
successive proteolytic “clips” to a form that exhibits the
characteristic activity of the mature protein, eg, its
enzymatic activity. Proteins synthesized as proproteins include the hormone insulin (proprotein
= proinsulin), the digestive enzymes pepsin, trypsin, and chymotrypsin
(proproteins = pepsinogen, trypsinogen, and
chymotrypsinogen, respectively), several factors of the blood
clotting and blood clot dissolution cascades , and
the connective tissue protein collagen (proprotein =
procollagen).
Proenzymes
Facilitate Rapid Mobilization
of an Activity in Response
to
Physiologic Demand
The synthesis and secretion of proteases as catalytically inactive
proenzymes protects the tissue of origin (eg, the
pancreas) from autodigestion, such as can occur in pancreatitis.
Certain physiologic processes such as digestion are
intermittent but fairly regular and predictable. Others
such as blood clot formation, clot dissolution, and
tissue repair are brought “on line” only in
response to pressing physiologic or pathophysiologic need.
The processes of blood clot formation and dissolution clearly
must be temporally coordinated to
achieve homeostasis. Enzymes needed intermittently but
rapidly often are secreted in an initially inactive form
since the secretion process or new synthesis of the required
proteins might be insufficiently rapid for response to a
pressing pathophysiologic demand such as the loss of
blood.
Activation of Prochymotrypsin Requires Selective Proteolysis
Selective
proteolysis involves one or more highly specific proteolytic
clips that may or may not be accompanied by separation of
the resulting peptides. Most importantly, selective
proteolysis often results in conformational
changes that “create” the catalytic site of an enzyme.
Note that while His 57 and Asp 102 reside on the B peptide
of α-chymotrypsin, Ser 195 resides on the C
peptide. The conformational changes that accompany
selective proteolysis of prochymotrypsin
(chymotrypsinogen) align the three residues of the
charge-relay network, creating the catalytic site.
Note also that contact and catalytic residues can be
located on different peptide chains but still be within
bond-forming distance of bound substrate.
REVERSIBLE
COVALENT MODIFICATION REGULATES KEY MAMMALIAN ENZYMES
Mammalian proteins are the targets of a wide range of covalent
modification processes. Modifications such as glycosylation,
hydroxylation, and fatty acid acylation introduce new
structural features into newly synthesized proteins
that tend to persist for the lifetime of the protein.
Among the covalent modifications that regulate protein
function (eg, methylation, adenylylation), the most
common by far is phosphorylation-dephosphorylation.
Protein kinases phosphorylate proteins by catalyzing
transfer of the terminal phosphoryl group of ATP to
the hydroxyl groups of seryl, threonyl, or tyrosyl
residues, forming O-phosphoseryl, O-phosphothreonyl, or O-phosphotyrosyl
residues, respectively. Some protein kinases target the side chains of
histidyl, lysyl, arginyl, and aspartyl residues.
The unmodified form of the protein can be regenerated
by hydrolytic removal of phosphoryl groups, catalyzed
by protein phosphatases.
A typical mammalian cell possesses over 1000 phosphorylated
proteins and several hundred protein kinases and
protein phosphatases that catalyze their interconversion. The ease
of interconversion of enzymes between
their phospho- and dephospho- forms in part dephosphorylation as a
mechanism for regulatory control. Phosphorylation-dephosphorylation
permits the functional properties of the affected enzyme to be
altered only for as long as it serves a specific
need. Once the need has passed, the enzyme can be
converted back to its original form, poised to respond to
the next stimulatory event. A second factor underlying the
widespread use of protein
phosphorylation-dephosphorylation lies in the chemical properties
of the phosphoryl group itself.
In order to alter an enzyme’s functional properties, any
modification of its chemical structure must influence the
protein’s three-dimensional configuration.
The high charge density of protein-bound phosphoryl groups—generally
−2 at physiologic pH—and their propensity to
form salt bridges with arginyl residues
make them potent agents for modifying protein structure and
function. Phosphorylation generally targets amino
acids distant from the catalytic site itself. Consequent conformational
changes then influence an enzyme’s
intrinsic catalytic efficiency or other properties. In this
sense, the sites of phosphorylation and other covalent modifications
can be considered another form of allosteric site.
However, in this case the “allosteric ligand” binds
covalently to the protein.
PROTEIN PHOSPHORYLATION IS EXTREMELY VERSATILE
Protein
phosphorylation-dephosphorylation is a highly versatile
and selective process. Not all proteins are subject to
phosphorylation, and of the many hydroxyl groups on a
protein’s surface, only one or a small subset are
targeted. While the most common enzyme function affected
is the protein’s catalytic efficiency, phosphorylation can also
alter the affinity for substrates, location
within
the cell, or responsiveness to regulation by allosteric ligands.
Phosphorylation can increase an enzyme’s catalytic
efficiency, converting it to its active form in one
protein, while phosphorylation of another converts it into
an intrinsically inefficient, or inactive, form.
Many
proteins can be phosphorylated at multiple sites or
are subject to regulation both by phosphorylation-dephosphorylation and by the
binding of allosteric ligands.
Phosphorylation-dephosphorylation at any one site can
be catalyzed by multiple protein kinases or protein phosphatases.
Many protein kinases and most protein phosphatases act
on more than one protein and are
themselves
interconverted between active and inactive forms by
the binding of second messengers or by covalent modification
by phosphorylation-dephosphorylation. The interplay
between protein kinases and protein phosphatases,
between the functional consequences of phosphorylation
at different sites, or between phosphorylation sites and
allosteric sites provides the basis for regulatory
networks that integrate multiple environmental input
signals to evoke an appropriate coordinated cellular
response. In these sophisticated regulatory networks,
individual enzymes respond to different environmental
signals. For example, if an enzyme can be
phosphorylated at a single site by more than one protein
kinase, it can be converted from a catalytically efficient
to an inefficient (inactive) form, or vice versa, in
response to any one of several signals. If the protein kinase
is activated in response to a signal different from the
signal that activates the protein phosphatase, the phosphoprotein
becomes a decision node. The functional output,
generally catalytic activity, reflects the phosphorylation
state. This state or degree of phosphorylation is
determined by the relative activities of the protein
kinase and protein phosphatase, a reflection of the
presence and relative strength of the environmental signals
that act through each. The ability of many protein
kinases and protein phosphatases to target more
than one protein provides a means for an environmental signal
to coordinately regulate multiple metabolic
processes. For example, the enzymes 3-hydroxy- 3-methylglutaryl-CoA
reductase and acetyl-CoA carboxylase—the
rate-controlling enzymes for cholesterol and fatty acid
biosynthesis, respectively—are phosphorylated
and inactivated by the AMP-activated protein kinase.
When this protein kinase is activated either through
phosphorylation by yet another protein kinase or in
response to the binding of its allosteric activator 5′-AMP,
the two major pathways responsible for the synthesis of
lipids from acetyl-CoA both are inhibited. Interconvertible
enzymes and the enzymes responsible for their
interconversion do not act as mere on and off switches
working independently of one another.
Enzymes are biological catalysts
responsible for supporting almost all of the chemical reactions that maintain
animal homeostasis. Because of their role in maintaining life processes, the
assay and pharmacological regulation of enzymes have become key elements in
clinical diagnosis and therapeutics. The macromolecular components of almost
all enzymes are composed of protein, except for a class of RNA modifying
catalysts known as ribozymes. Ribozymes are molecules of ribonucleic acid that
catalyze reactions on the phosphodiester bond of other RNAs.
Enzymes
are found in all tissues and fluids of the body. Intracellular enzymes catalyze
the reactions of metabolic pathways. Plasma membrane enzymes regulate catalysis
within cells in response to extracellular signals, and enzymes of the
circulatory system are responsible for regulating the clotting of blood. Almost
every significant life process is dependent on enzyme activity.
http://www.youtube.com/watch?v=Ofs0mfkl370
Enzyme Classifications
Traditionally, enzymes were simply
assigned names by the investigator who discovered the enzyme. As knowledge
expanded, systems of enzyme classification became more comprehensive and
complex. Currently enzymes are grouped into six functional classes by the
International Union of Biochemists (I.U.B.).
Number |
Classification |
Biochemical Properties |
1. |
Oxidoreductases |
Act on many chemical groupings to
add or remove hydrogen atoms. |
2. |
Transferases |
Transfer functional groups between
donor and acceptor molecules. Kinases are specialized transferases that
regulate metabolism by transferring phosphate from ATP to other molecules. |
3. |
Hydrolases |
Add water across a bond, hydrolyzing
it. |
4. |
Lyases |
Add water, ammonia or carbon
dioxide across double bonds, or remove these elements to produce double
bonds. |
5. |
Isomerases |
Carry out many kinds of isomerization:
L to D isomerizations, mutase reactions (shifts of chemical groups) and
others. |
6. |
Ligases |
Catalyze reactions in which two
chemical groups are joined (or ligated) with the use of energy from ATP. |
These rules give each enzyme a unique
number. The I.U.B. system also specifies a textual name for each enzyme. The
enzyme's name is comprised of the names of the substrate(s), the product(s) and
the enzyme's functional class. Because many enzymes, such as alcohol
dehydrogenase, are widely known in the scientific community by their common
names, the change to I.U.B.-approved nomenclature has been slow. In everyday
usage, most enzymes are still called by their common name.
Enzymes are also classified on the
basis of their composition. Enzymes composed wholly of protein are known as
simple enzymes in contrast to complex enzymes, which are composed of protein
plus a relatively small organic molecule. Complex enzymes are also known as
holoenzymes. In this terminology the protein component is known as the
apoenzyme, while the non-protein component is known as the coenzyme or
prosthetic group where prosthetic group describes a complex in which the small
organic molecule is bound to the apoenzyme by covalent bonds; when the binding
between the apoenzyme and non-protein components is non-covalent, the small
organic molecule is called a coenzyme. Many prosthetic groups and coenzymes are
water-soluble derivatives of vitamins. It should be noted that the main
clinical symptoms of dietary vitamin insufficiency generally arise from the
malfunction of enzymes, which lack sufficient cofactors derived from vitamins
to maintain homeostasis.
The non-protein component of an
enzyme may be as simple as a metal ion or as complex as a small non-protein
organic molecule. Enzymes that require a metal
in their composition are known as metalloenzymes if they bind and retain their
metal atom(s) under all conditions with very high affinity. Those which have a
lower affinity for metal ion, but still require the metal ion for activity, are
known as metal-activated enzymes.
Enzymes in
the Diagnosis of Pathology
http://www.youtube.com/watch?v=5hrU6_tic7s&feature=related
The measurement of the serum levels
of numerous enzymes has been shown to be of diagnostic significance. This is
because the presence of these enzymes in the serum indicates that tissue or
cellular damage has occurred resulting in the release of intracellular
components into the blood. Hence, when a physician indicates that he/she is
going to assay for liver enzymes, the purpose is to ascertain the potential for
liver cell damage. Commonly assayed enzymes are the amino transferases: alanine
transaminase, ALT (sometimes still referred to as serum glutamate-pyruvate
aminotransferase, SGPT) and aspartate aminotransferase, AST (also referred to
as serum glutamate-oxaloacetate aminotransferase, SGOT); lactate dehydrogenase,
LDH; creatine kinase, CK (also called creatine phosphokinase, CPK);
gamma-glutamyl transpeptidase, GGT. Other enzymes are assayed under a variety
of different clinical situations but they will not be covered here.
The typical liver
enzymes measured are AST and ALT. ALT is particularly diagnostic of liver
involvement as this enzyme is found predominantly in hepatocytes. When assaying
for both ALT and AST the ratio of the level of these two enzymes can also be
diagnostic. Normally in liver disease or damage that is not of viral origin the
ratio of ALT/AST is less than 1. However, with viral hepatitis the ALT/AST
ratio will be greater than 1. Measurement of AST is useful not only for liver
involvement but also for heart disease or damage. The level of AST elevation in the
serum is directly proportional to the number of cells involved as well as on
the time following injury that the AST assay was performed. Following injury,
levels of AST rise within 8 hours and peak 24-36 hours later. Within 3-7 days
the level of AST should return to pre-injury levels, provided a continuous
insult is not present or further injury occurs. Although measurement of AST is
not, in and of itself, diagnostic for myocardial infarction, taken together
with LDH and CK measurements (see below) the level of AST is useful for timing
of the infarct.
The measurement of LDH is especially
diagnostic for myocardial infarction because this enzyme exist in 5 closely
related, but slightly different forms (isozymes). The 5 types and their normal
distribution and levels in non-disease/injury are listed below.
LDH 1 - Found in heart and red-blood
cells and is 17% - 27% of the normal serum total.
LDH 2 - Found in heart and red-blood
cells and is 27% - 37% of the normal serum total.
LDH 3 - Found in a variety of organs
and is 18% - 25% of the normal serum total.
LDH 4 - Found in a variety of organs
and is 3% - 8% of the normal serum total.
LDH 5 - Found in liver and skeletal
muscle and is 0% - 5% of the normal serum total.
Following a
myocardial infarct the serum levels of LDH rise within 24-48 hours reaching a
peak by 2-3 days and return to normal in 5-10 days. Especially diagnostic is a
comparison of the LDH-1/LDH-2 ratio. Normally, this ration is less than
CPK is found
primarily in heart and skeletal muscle as well as the brain. Therefore,
measurement of serum CPK levels is a good diagnostic for injury to these
tissues. The levels of CPK will rise within 6 hours of injury and peak by around
18 hours. If the injury is not persistent the level of CK returns to normal
within 2-3 days.
Like LDH, there are tissue-specific isozymes of CPK and there designations are
described below.
CPK3 (CPK-MM) is the predominant
isozyme in muscle and is 100% of the normal serum total.
CPK2 (CPK-MB) accounts for about 35%
of the CPK activity in cardiac muscle, but less than 5% in skeletal muscle and
is 0% of the normal serum total.
CPK1 (CPK-BB) is the characteristic
isozyme in brain and is in significant amounts in smooth muscle and is 0% of
the normal serum total.
Since most of the released CPK after
a myocardial infarction is CPK-MB, an increased ratio of CPK-MB to total CPK
may help in diagnosis of an acute infarction, but an increase of total CPK in
itself may not. CPK-MB levels rise 3-6 hours after a myocardial infarct and
peak 12-24 hours later if no further damage occurs and returns to normal 12-48
hours after the infarct.
CLINICAL ENZYMOLOGY
http://www.youtube.com/watch?v=5hrU6_tic7s&feature=related
PLASMA
ENZYMES
Measurements
of the activity of enzymes in plasma are of value in the diagnosis and
management of a wide variety of diseases. Most enzymes measured in plasma are
primarily intracellular, being released into the blood when there is damage to
cell membranes, but many enzymes, for example renin, complement factors and
coagulation factors, are actively secreted into the blood, where they fulfil
their physiological functions. Small amounts of intracellular enzymes are
present in the blood as a result of normal cell turnover. When damage to cells
occurs, increased amounts of enzymes will be released and their concentrations
in the blood will rise. However, such increases are not always due to tissue
damage. Other possible causes include:
·
increased cell turnover
·
cellular proliferation (e.g. neoplasia)
·
increased enzyme synthesis (enzyme induction)
·
obstruction to secretion
·
decreased clearance.
Little is known about the mechanisms
by which enzymes are removed from the circulation. Small molecules, such as
amylase, are filtered by the glomeruli but most enzymes are probably removed by
reticuloendothelial cells. Plasma amylase activity rises in acute renal failure
but, in general, changes in clearance rates are not known to be important as
causes of changes in plasma enzyme levels.
Plasma
contains many functional enzymes, which
a actively secreted into plasma. For example, enzymes blood coagulation. On the
other hand, there are a few non functional enzymes in plasma, which are coming out from cells of various tissues due
to normal wear and tear. Their normal levels in blood are very low; but are
drastically increased during cell death (necrosis) or disease. Therefore assays
of these enzymes are very useful in diagnosis diseases.
Enzyme assays usually depend on the
measurement the catalytic activity of the enzyme, rather than the concentration
of the enzyme protein itself. Since each enzyme molecule can catalyze the
reaction of many molecules of substrate, measurement of activity provides great
sensitivity. It is, however, important that the conditions of the assay are
optimized and standardized to give reliable and reproducible results. Reference
ranges for plasma enzymes are dependent on assay conditions, for example
temperature, and may also be subject to physiological influences. It is thus
important to be aware of both the reference range for the laboratory providing
the assay and the physiological circumstances when interpreting the results of
enzyme assays.
One
international unit is
the amount of enzyme that will convert one micromole of substrate per minute
per litre of sample and is abbreviated as U/L. The SI Unit (System Internationale)
expression is more scientific, where or Katal
(catalytic activity) is defined as the number of mole of substrate
transformed per second per litre of sample. Katal is abbreviated as kat or k
(60 U = 1 μkat and 1 nk = 0.06 U).
Disadvantages of enzyme assays
A major disadvantage in the use of
enzymes for the diagnosis of tissue damage is their lack of specificity to a
particular tissue or cell type. Many enzymes are common to more than one
tissue, with the result that an increase in the plasma activity of a particular
enzyme could reflect damage to any one of these tissues. This problem may be
obviated to some extent in two ways:
first,
different tissues may contain (and thus release when they are damaged) two or
more enzymes in different proportions; thus alanine and aspartate
aminotransferases are both present in cardiac and skeletal muscle and
hepatocytes, but there is only a very little alanine aminotransferase in either
type of muscle;
second,
some enzymes exist in different forms (isoforms), colloquially termed
isoenzymes (although, strictly, the term 'isoenzyme' refers only to a
genetically determined isoform). Individual isoforms are often characteristic
of a particular tissue: although they may have similar catalytic activities,
they often differ in some other measurable property, such as heat stability or
sensitivity to inhibitors.
After a single insult to a tissue,
the activity of intracellular enzymes in the plasma rises as they are released
from the damaged cells, and then falls as the enzymes are cleared. It is thus
important to consider the time at which the blood sample is taken in relation
to the insult. If taken too soon, there may have been insufficient time for the
enzyme to reach the blood- stream and if too late, it may have been completely
cleared. As with all diagnostic techniques, data acquired from measurements of
enzymes in plasma must always be assessed in the light of whatever clinical and
other information is available, and their limitations borne in mind.
LACTATE
DEHYDROGENASE (LDH) (LD)
The total LDH is generally tested by
reaction of the serum sample with pyruvate and NADH2. LDH will
convert pyruvate to lactate, and in turn NADH is use up by the reaction.
Normal value of LDH in serum is 100-200 U/L.
Values the upper range are generally seen in children. Strenuous exercise will
slightly increase the value. LDH level is 100 times more inside the RBC than in
plasma, and therefore minor amount of hemolysis will result in a false-positive
test.
LDH and Heart Attack
In
myocardial infarction, total LDH activity is increased, while H4
iso-enzyme is increased 5-10 times more.
Differential
diagnosis: Increase
in total LDH level is seen in
hemolytic anemias, hepatocellular damage, muscular dystrophy, carcinomas,
leukemias, and any condition which causes necrosis of body cells. Since total
LDH is increased in many conditions, the study of isozymes of LDH is of great
importance.
Isoenzymes of LDH
LDH enzyme is a tetramer with four
subunits. But the subunit may be either H (heart) or M (muscle) polypeptide
chains. These two are the products of two different genes.
Although both of them have the same
molecular weight (32 kD), there are minor amino acid variations. So five
combinations of H and M chains are possible; H4, H3M, H2M2,
M3H and M4 varieties, forming five iso-enzymes.
All these five forms are seen in all persons.
M4 form is seen in skeletal muscles; it is not inhibited by
pyruvate. But H4 form is seen in heart and is inhibited by pyruvate.
Normally LDH-2 (H3M1) concentration in blood is greater
than LDH-1 (H4); but this pattern is reversed in myocardial infarction; this is
called flipped pattern. The
iso-enzymes are usual ly separated by cellulose acetate electrophoresis at pH 8.6. They are then identified by adding the
reactants finally producing a colour reaction. . Lactate dehydrogenase
isoenzymes (as percentage of total):
LDH1 14-26 %
LDH2 29-39 %
LDH3 20-26 %
LDH4 8-16%
LDH5 6-16 %
CREATINE KINASE(CK)
http://www.youtube.com/watch?v=6r5Ddlcq26s
Creatine
→ Creatine phosphate
It
was called as creatine phosphokinase in old literature.
Normal serum value for CK is 15-100 U/L for males
and 10-80 U/L for females.
CK
and Heart Attack
CK value in serum is increased in myocardial infarction. The CK level starts to rise within three hours of infarction. Therefore, CK estimation is very useful to detect
early cases, where ECG changes may be ambiguous. The CK level is not increased
in hemolysis or in congestive cardiac failure; and therefore CK has an
advantage over LDH.
CK
and Muscle Diseases
he level of CK in serum is very much elevated in muscular dystrophies (500
-1500 IU/L). The level is very high in the early phases of the disease. In such
patients a fall in CK level is indicative of deteriorating condition, because
by that time, all muscle mass is destroyed. In female carriers of this X-linked
disease (genotypically heterozygous), CK is seen to be moderately raised. CK
level is highly elevated in crush injury, fracture and acute cerebrovascular
accidents. Estimation of total CK is
employed in muscular dystrophies and MB iso-enzyme is estimated in myocardial
infarction.
Iso-enzymes
of CK CK is a
dimer; each subunit has a molecular weight of 40,000. The subunits are called B
for brain and M for muscle. They are products of loci in chromosomes 14 and 19
respectively. Therefore three iso-enzymes are seen in circulation. Normally CK2
is only 5% of the total activity. Even doubling the value in CK2 (MB)
iso-enzyme may not be detected, if total value of CK alone is estimated. Hence
the detection of MB-iso-enzyme is
important in myocardial infarction. CK-MB < 6 % of total CK in normal
conditions.
The above three iso-enzymes are
cytosolic. A fourth variety, called CK-mt is located in mitochondria and
constitutes about 15% of total CK activity. Its gene is located in chromosome
15. CK1 may be complexed with immunoglobulin; and then termed macroCK. CK1-lgG
causes false-positive diagnosis of myocardial infarction because it has an
electrophoretic mobility close to CK2.
For
quantitating MB iso-enzyme, anti-MM antiserum is added to the patient's serum.
This will precipitate MM iso-enzyme. The supernatant serum is used for the CK
estimation. Here it is assumed that BB isoenzyme is negligible in quantity,
which is correct if there is no brain disease. CK iso-enzymes can also be
identified by electrophoresis.
ASPARTATE AMINO TRANSFERASE (AST)
It is also called as serum
glutamate-oxaloacetate transaminase (SGOT). AST needs pyridoxal phosphate as
co-enzyme. AST is estimated by taking aspartate, α-ketoglutarate, pyridoxal phosphate
(vitamin B6) and patient' serum as the source of AST. The
oxaloacetate formed may be allowed to react with dinitrophenyl hydrazine to produce
a colour which is estimated colorimetrically at 520 nm.
Normal
serum level of AST is 8-40 U/L or (0,1-0,45 mmol/(hour´L))
It is significantly elevated in myocardial infarction. It if moderately elevated in liver diseases.
However, a marked increase in AST may be seen in primary hepatoma. AST has two
iso-enzymes; cytoplasmic and mitochondrial. In mile degree of tissue injury,
cytoplasmic form is seen in serum. Mitochondrial type is seen in severe injury.
ALANINE AMINO TRANSFERASE (ALT)
It is also called as serum
glutamate-pyruvate transaminase (SGPT). ALT needs pyridoxal phosphate as
co-enzyme.
Normal
serum level of ALT is 5-30 U/L or (0,1-0,68 mmol/(hour´L))
Very
high values (100 to 1000 U/L) are seen in acute hepatitis, either toxic or
viral in origin. Both ALT and AST are increased in liver diseases, but ALT
>AST. Moderate increase (25 to 100 U/L) may be seen in chronic liver disease
such as cirrhosis, and malignancy in liver. A sudden fall in ALT level in cases
of hepatitis is a very bad prognostic sign.
Ritis
coefficient (AST/ALT) in normal conditions is 1,33±0,42.
http://www.youtube.com/watch?v=nXRWkorYFXc
Post mortem
specimen of cirrhotic liver and enlarged spleen
Alkaline
phosphatase (ALP)
It is a non- specific enzyme which
hydrolyses aliphatic, aromatic or heterocyclic compounds. The pH optimum for
the enzyme reaction is between 9 and 10. It is prodused by osteoblasts of bone,
and localized in cell memmbranes (ecto-enzyme).
Normal serum level of ALP is 40-125
U/L or 0,5-1,3 mmol/(hour´ L).
In children the upper level of normal
value may be more, becouse of the increased osteoblastic activity. Mild increase
is noticed during pregnancy, due to production of placental isoenzyme.
Moderate (2-3 times) increase in ALP
level is seen in hepatic diseases such as hepatitis, alcoholic hepatosis or
hepatocellular carcinoma. Very high levels of ALP (10-12 times of upper limit)
may be noticed in extrahepatic obstructions or cholestasis. ALP is produced by
epithelial cells of biliary canaliculi and obstruction of bile with consequent
irritation of epithelial cells leads to secretion of ALP into serum.
Drastically high levels of ALP (10-25
times of upper limit) are also seen in bone diseases where osteoblastic
activity is enhanced such as Paget's disease, rickets, osteomalacia,
osteoblastoma, metastatic carcinoma of bone and hyperparathyroidism (Paget's disease
or osteitis deformans was described in 1877 by Sir James Paget).
Iso-enzymes
of Alkaline Phosphatase
1. α-1 ALP moves in α -1 position, it is synthesised by
epithelial cells of biliary canaliculi. It is about 10% of total activity and
is increased in obstructive jaundice and to some extent in metastatic carcinoma
of liver.
2. α -2 heat labile ALP is stable at
3. α -2 heat stable ALP will not be
destroyed at
4. Pre-ß ALP is of bone origin
and elevated levels are seen in bone diseases. This is the most heat labile
(destroyed at
5. γ-ALP is inhibited by phenylalanine
and originates from intestinal cells. It is increased in ulcerative colitis.
About 10% of plasma ALP are of intestinal variety.
6. The leucocyte alkaline phosphatase
(LAP) is significantly decreased in chronic myeloid leukemia. It is increased
in lymphomas.
ALP has different isoforms. Although
ALP is a monomer, depending on the number of sialic acid residues, the charged
groups differ. Such different forms are detected in agar gel electrophoresis.
NUCLEOTIDE PHOSPHATASE (NTP)
It is also known as 5' nucleotidase.
This enzyme hydrolyses 5' nucleotides to corresponding nucleosides at an
optimum pH of 7.5. It is a marker enzyme for plasma membranes and is seen as an
ecto-enzyme (enzyme present on the cell membrane).
Usually, AMP is used as substrate,
which is hydrolysed to adenosine and inorganic phosphate. The latter reacts
with ammonium molybdate to produce the yellow ammonium phosphomolybdate, which
is estimated colorimetrically. However, ALP will also catalyse the same
reaction. Serum samples contain both ALP and NTP. These are distinguished by
Nickel ions which inhibit NTP but not ALP.
Normal
NTP level in serum is 2-10 U/L. It is moderately increased in hepatitis and
highly elevated in biliary obstruction. Unlike ALP, the level is unrelated with
osteoblastic activity and therefore is unaffected by bone diseases.
GAMMA
GLUTAMYL TRANSFERASE (GGT)
The old name was gamma glutamyl
transpeptidase. It can transfer γ-glutamyl residues to substrate. In
the body it is used in the synthesis of glutathione. GGT has 11 iso-enzymes. It
is seen in liver, kidney, pancreas, intestinal cells and prostate gland.
Normal serum value of GGT is 6-45 U/L
in male and 5-30 U/L in female. It is slightly higher in normal males, due to
the presence of prostate gland. This value is moderately increased in infective
hepatitis and prostate cancers. The GGT level is highly elevated in alcoholism,
obstructive jaundice and neoplasm's of liver. GGT-2 is positive for 90% of
hepatocellular carcinomas. It is not elevated in cardiac or skeletal diseases.
GGT
is a microsomal enzyme. Its activity is induced by alcohol, phenobarbitone and
rifampicin. GGT is clinically important because of its sensitivity to detect
alcohol abuse. GGT is increased in alcoholics even when other liver function
tests are within normal limits. GGT level is rapidly decreased within a few
days when the person stops to take alcohol. Increase in GGT level is generally
proportional to the amount of alcohol intake.
ACID
PHOSPHATASE (ACP)
It hydrolyses phosphoric acid ester
at pH between 4 and 6. Methods for assay are the same as described for ALP; but
the pH of the medium is kept at 5 to 5.4.
Normal serum value for ACP is 2.5-12
U/L or 0,025-0,12 mmol/(hour´ L).
ACP is secreted by prostate cells,
RBC, platelets and WBC. Isoenzymes of ACP are described. Erythrocyte ACP gene
is located in chromosome 2; osteoclast ACP gene is on chromosome 19; lysosomal
gene is on 11 and prostate ACP gene is on 13. The prostate iso-enzyme is
inactivated by tartaric acid. Cupric ions inhibit erythrocyte ACP. Normal level
of tartrate labile fraction of ACP is 1 U/L.
ACP total value is increased in
prostate cancer and highly elevated in bone metastasis of prostate cancer. In
these conditions, the tartrate labile iso-enzyme is elevated. This assay is
very helpful in follow up of treatment of prostate cancers. ACP is therefore an important tumour marker.
Since blood cells
contain excess quantity of ACP, must be taken to prevent hemolysis while taking
blood from the patient. Prostate massage may also increase to value. So blood
may be collected for ACP estimation before per rectal examination of patient.
ACP is present in high concentration in semen, a finding which is used in
forensic medicine in investigation of rape.
PROSTATE
SPECIFIC ANTIGEN (PSA)
It is produced from the secretory
epithelium of prostal gland. It is normally secreted into seminal fluid, where
it is necessary for the liquefaction of seminal coagulum. It is a serine
protease, and is a 32 kD glycoprotein; encoded in chromosome number
Normal
value is 1 -5 µg/L. It is very specific for prostate activity. Values between
4-10 µg/L is seen in benign prostate enlargement; but values above 10 µg/L is
indicative of prostate cancer.
CHOLINESTERASE
(ChE)
Acetyl cholinesterase or true ChE or
Type 1 ChE can act mainly on acetyl choline. It is present in nerve endings and
in RBCs. About 25 allelic forms are reported. Normal serum range is 2-12 U/ml.
Newly formed RBC will contain good quantity of ChE which is slowly reduced
according to the age of the cell. Therefore, ChE level in RBCs will be
proportional to the reticulocyte count. Organophosphorus insecticides
(Parathione) irreversibly inhibit ChE in RBCs. Measurement of ChE level in RBCs
is useful to determine the amount of exposure in persons working with these
insecticides.
Pseudocholinesterase or type II ChE
is non-specific and can hydrolyse acyl esters. It is produced mainly by liver
cells. Normal serum level is 8-18 U/ml. Succinyl choline is a widely used as
muscle relaxant. It is a structural analogue of ACh, and so competitively fix
on post-synaptic receptors of ACh. Succinyl choline is hydrolysed by the liver
ChE within 2-4 minutes. But in certain persons the ChE activity may be absent;
this is a genetically transmitted condition. In such individuals when succinyl
choline is given during surgery, it may take hours to get the drug metabolised.
Very prolonged scoline apnoea may result in 'nightmare of anaesthetist'. The
pseudocholinesterase level in serum is reduced in viral hepatitis, cirrhosis,
hepatocellular carcinoma, metastatic cancer of liver and in malnutrition.
http://www.youtube.com/watch?v=-gIqZ8IxctE
GLUCOSE-6-PHOSPHATE
DEHYDROGENASE
GPD is a dimer with identical
subunits. This is an important enzyme in the hexose monophosphate shunt pathway
of glucose. It is mainly used for production of NADPH . It has a special role
in the RBC metabolism. Due to the presence of oxygen, hydrogen peroxide is
continuously formed inside the RBC. Peroxide will destroy biomembranes, and
RBCs are lysed. Normal value of GPD in RBC is 125-250 U/1012 cells.
Nearly 400 variants (isoforms) of GPD are described.
AMYLASE
This splits starch to maltose. It is
activated by calcium, chloride and fluoride ions. There are 18 phenotypes. It
is produced by pancreas and salivary glands; they are products of different
genes located in chromosome 1.
http://www.youtube.com/watch?v=AEsQxzeAry8
Normal
serum value is 50-120 U/L, (12-32 g/(hour× L)).
The value is increased about 1000
times in acute pancreatitis which is a life-threatening condition. The peak
values are seen between 5-12 hours after the onset of disease and returns to
normal levels within 2-4 days after the acute phase has subsided. Moderate increase in serum levels are seen in
chronic pancreatitis, mumps (parotitis), obstruction of pancreatic duct and in
renal disease. In the last condition, the enzyme is not excreted through urine
properly and hence serum value is raised. Normal urine value is 20-160
g/(hour× L) or (less than 375 U/L). It is increased in acute
pancreatitis. It is increased on the
LIPASE
It will hydrolyse triglyceride to β-monoglyceride and fatty acid.
Molecular weight is 54,000. The gene is in chromosome 10. The enzyme is present
in pancreatic secretion. Normal serum range is 0.2-1.5 U/L. It is highly
elevated in acute pancreatitis and this persists for 7-14 days. Thus, lipase
remains elevated longer than amylase. Moreover, lipase is not increased in
mumps. Therefore, lipase estimation has advantage over amylase. It is
moderately increased in carcinoma of pancreas, biliary diseases and perforating
peptic ulcers.
ALDOLASE (ALD)
It is a tetrameric
enzyme with A and B subunits; so there are 5 iso-enzymes. It is a glycolytic
enzyme. Normal range of serum is 1.5-7 U/L. It is drastically elevated in
muscle damages such as progressive muscular dystrophy, poliomyelitis,
myasthenia gravis and multiple sclerosis. It is a very sensitive early index in
muscle wasting diseases.
ENOLASE
It is a glycolytic enzyme.
Neuron-specific enolase (NSE) is an iso-enzyme seen in neural tissues and
Apudomas. NSE is a tumour marker for cancers associated with neuro-endocrine
origin, small cell lung cancer, neuroblastoma, pheochromocytoma, medullary
carcinoma of thyroid, etc. It is measured by RIA or ELISA. Upper limit of NSE
is 12 μg/ml.
Usage enzymes in medical practice
Enzymo-pathologies – disoders of enzymes action. Enzymo-pathologies takes place
actually at any disease (it’s so called secondary enzymo-pathologies). But
there also are more than one thousand different forms of congenital molecular
pathologies (primary enzymo-pathologies). Mutations (genetic disorders) were
found to be the causes of these kind of enzymo-pathologies.
The
examples of primary enzymo-pathologies:
1) Enzyme phenylalanine
4-monooxygenase is absent in about
Alloenzyme - Isoenzyme
another pathway phenylalanine undergoes transamination with alpha-ketoglutarate
to yield phenylpyruvic acid, which accumulates in the blood and is excreted in
the urine. In childhood excess circulating phenylpyruvate impairs normal brain
development, causing severe mental retardation. This condition, phenylketonuria, was among the first
genetic defects of metabolism recognized in man. Restriction of dietary
phenylalanine during childhpood prevents the mental retardation.
2) The absence of homogentisic
acid 1,2-dioxygenase causes urinary excretion of phenylpyruvic and
homogentisic acids. The urinary of people genetically defective in homogentisic
acid 1,2-dioxygenase contains homogentisic acid, which when made alkaline and
exposed to oxygen, turns dark because it is oxidized and polimerized to a black
melanin pigment. This condition is known as alkaptonuria.
Patients with this condition have abnormal pigmentation of the connective
tissue.
3) D-galactose is converted into D-glucose in the liver by
special reactions which have attracted much attention because they are subject
to genetic defects in man, resulting in different forms of the hereditary
disease galactosemia. The deffect or
absence of enzyme galactose 1-phosphate
uridylyltransferase causes the increase of D-galactose in the blood.
Galactose 1-phosphate uridylyltransferase
is present in normal fetal liver but is lacking in infants with
galactosemia. Galactosemic infants suffer from cataract of the lens of the eye
as well as mental disorders. This condition can be successfully treated by
withholding milk and other sources of galactose from the diet during infancy
and childhood.
Enzymo-therapy. In digestive tract
diseases (deficit of digestive enzymes) pepsin and HCI, enzymes of pancreas
(pancreatin, oraza, panzinorm) are recomended to use.
Proteolytic enzymes
trypsin and chemotrypsin are used for processing of wounds in burns, ulcers for
the a proteolysis and deleting of necrotic tissues.
Trypsin, ribonuclease, DNA-ase are used for the
splitting and deleting of fibrin from the pleural cavity, for the dilution and
deleting of sputum from the respiratory ways in acute and chronic respiratory
diseases, for the thrombophlebitis treatment.
Fibrinolysin, streptokinase are used for the
splitting of thrombus.
Hyaluronidase (lidase) is used for the acceleration of
different medicines penetration into the biological tissues as well as for the resorption of scars and
hematoma.
Cytochrome C is used in the intoxication of CO, H2S
and other substances oppressing the tissue respiration.
Glucoso-oxidase is used for
the lavage of wounds and burns as antiseptic.
Thrombin is used for
preventing and stop of bleeding.
The using of coenzymes. ATP is
used in heart diseases, muscle dystrophia.
TPP (thiamin pyrophosphate) is
used in heart diseases, and nervous system pathology.
FMN (flavinmononucleotide) is
used for treatment of skin diseases, keratitis, conjunctivitis.
NAD and NADP are used for
improvement of oxidative-reduction processes in organism.
The using of inhibitors of enzymes.
Inhibitors of proteolytic enzymes (kontrical, trasilol) are used in acute
pancreatitis for inhibition trypsin, chemotrypsin activity. Irreversible inhibitors
Some enzyme inhibitors react with the enzyme and form a covalent adduct with
the protein. The inactivation produced by this type of inhibitor is
irreversible. A class of these compounds called suicide inhibitors includes
eflornithine a drug used to treat the parasitic disease sleeping sickness.
Penicillin and its derivatives also act in this manner. With these drugs, the
compound is bound in the active site and the enzyme then converts the inhibitor
into an activated form that reacts irreversibly with one or more with amino
acid residues.
The coenzyme folic acid (left) and the anti-cancer drug methotrexate
(right) are very similar in structure. As a result, methotrexate is a
competitive inhibitor of many enzymes that use folates.
Uses
of inhibitors
Inhibitors are often used
as drugs, but they can also act as poisons. However, the difference between a
drug and a poison is usually only a matter of amount, since most drugs are
toxic at some level, as Paracelsus wrote, "In all things there is a
poison, and there is nothing without a poison." Equally, antibiotics and
other anti-infective drugs are just specific poisons that can kill a pathogen
but not its host.
An example of an
inhibitor being used as a drug is aspirin, which inhibits the COX-1 and COX-2
enzymes that produce the inflammation messenger prostaglandin, thus suppressing
pain and inflammation. The poison cyanide is an irreversible enzyme inhibitor
that combines with the copper and iron in the active site of the enzyme
cytochrome c oxidase and blocks cellular respiration.
In many organisms
inhibitors may act as part of a feedback mechanism. If an enzyme produces too
much of one substance in the organism, that substance may act as an inhibitor
for the enzyme that produces it, causing production of the substance to slow
down or stop when there is sufficient amount. This is a form of negative
feedback.
Diacarb – inhibitor of carbohydrase. This enzyme
regulates the metabolism of Na+ and K+ in kidney
canaliculus and diuresis.
Isoenzymes
Another type of regulation of metabolic activity is
through the participation of isozymes,
multiple forms of a given enzyme that occur within a single species of
organism or even in a single cell. Such multiple forms can be detected and
separated by gel electrophoresis of cell extracts; since they are coded by different
genes, they differ in amino acid composition and thus in their isoelectric pH
values.
Lactate dehydrogenase, one of the first enzymes in
this class to be studied intensively, occurs as five different isozymes in the
tissues of vertebrates. All these isozymes have been isolated. They all
catalyze the same overall reaction, Lactate + NAD+ ® pyruvate + NADH + H+
in which NAD+ and NADH symbolize the oxidized and reduced
forms, respectively, of nicotinamide adenine dinucleotide. All five isozymes
have the same molecular weight, about 134,000, and all contain four
polypeptide chains, each of molecular weight 33,500. The five isozymes consist
of five different combinations of two different kinds of polypeptide chains,
designated A and B. The
isozyme predominating in skeletal muscle has four identical A chains and is
designated A4; another, which predominates in heart, has four
identical  chains and is designated B4. The other three isozymes have the
composition À3Â,
A2B2, and ÀÂ3. Single A and  chains have been isolated and found
to differ significantly in amino acid content and sequence. When single A and Â
chains, which are inactive, are mixed in appropriate proportions, all the
different isozymes of lactate dehydrogenase can be made to form spontaneously
in the test tube and all have full catalytic activity.
Genetic research indicates that the amino acid sequences of the two
different polypeptide chains A and  of lactate dehydrogenase are coded by two different genes.
The biosynthesis of the two types of chains and thus the relative amounts of
the lactate dehydrogenase isozymes present in a given cell are under genetic
regulation. Isozymes of lactate dehydrogenase exist in different proportions in
different tissues. Moreover, the relative proportions of the lactate
dehydrogenase isozymes in a tissue may change during embryological development.
They are also important in diagnosis of heart and liver disease.
Careful kinetic study of the lactate dehydrogenase isozymes has
revealed that although they all catalyze the same reaction, they differ in
their dependence on substrate concentration, particularly pyruvate, as well as
their Vmax values when pyruvate is the substrate. The isozyme A4, characteristic
of skeletal muscle and embryonic tissues, reduces pyruvate to lactate at a
relatively high rate. The B4 isozyme, characteristic of the heart
and other red muscles, reduces pyruvate at a relatively low rate. Moreover, the
dehydrogenation of lactate catalyzed by the B4 isozyme is strongly
inhibited by pyruvate. The other lactate dehydrogenase isozymes have kinetic
properties intermediate between those of the A4 and B4
isozymes, in proportion to their relative content of A and Â
chains.
Isozymes are now known for a great many different enzymes. They usually
consist of tightly associated mixtures of different kinds of polypeptide
chains, in which are blended the specific kinetic and binding properties
contributed by each type of chain. Many allosteric enzymes occur as two or more
isozymes that vary in sensitivity to their allosteric modulators. In other
cases different isozymes are present in different intracellular compartments.
The study of isozymes has
grown into one of fundamental significance in the investigation of the
molecular basis of cellular differentiation and morphogenesis. Many proteins in
cells, not only those with catalytic activity, may occur in multiple forms.
Enzyme cofactors
Many
enzymes require the presence of an additional, nonprotein, cofactor.
·
Some of these are metal ions
such as Zn2+ (the cofactor for carbonic anhydrase), Cu2+,
Mn2+, K+, and Na+.
·
Some cofactors are small
organic molecules called coenzymes.
The B vitamins
o
thiamine (B1)
o
riboflavin (B2) and
are precursors of coenzymes.
Coenzymes may be
covalently bound to the protein part (called the apoenzyme) of enzymes as a prosthetic
group. Others bind more loosely and, in fact, may bind only transiently
to the enzyme as it performs its atalytic
act.
Lysozyme: a model of enzyme
action
A
number of lysozymes are found in nature; in human tears and egg white, for
examples. The enzyme is antibacterial because it degrades the polysaccharide
that is found in the cell walls of many bacteria. It does this by catalyzing
the insertion of a water molecule at the position indicated by the red arrow.
This hydrolysis breaks the
chain at that point.
The
bacterial polysaccharide consists of long chains of alternating amino sugars:
·
N-acetylglucosamine (NAG)
·
N-acetylmuramic
acid (NAM)
These
hexose units resemble glucose except for the presence of the side chains
containing amino groups.
Lysozyme is a
globular protein with a deep cleft across part of its surface. Six hexoses of
the substrate fit into this cleft.
·
With so many oxygen atoms in
sugars, as many as 14 hydrogen bonds form
between the six amino sugars and certain amino acid R groups such as Arg-114, Asn-37, Asn-44, Trp-62, Trp-63, and Asp-101.
·
Some hydrogen bonds also
form with the C=O groups of several peptide bonds.
·
In addition, hydrophobic
interactions may help hold the substrate in position.
X-ray crystallography has shown that as
lysozyme and its substrate unite, each is slightly deformed. The fourth hexose
in the chain (ring #4) becomes twisted out of its normal position. This imposes
a strain on the C-O bond on the ring-4 side of the oxygen bridge between rings
4 and 5. It is just at this point that the polysaccharide is broken. A molecule
of water is inserted between these two hexoses, which breaks the chain. Here,
then, is a structural view of what it means to lower activation energy. The energy
needed to break this covalent bond is lower now that the atoms connected by the
bond have been distorted from their normal position.
As for lysozyme
itself, binding of the substrate induces a small (~0.75Å) movement of
certain amino acid residues so the cleft closes slightly over its substrate. So
the "lock" as well as the "key" changes shape as the two
are brought together. (This is sometimes called "induced fit".)
The amino acid
residues in the vicinity of rings 4 and 5 provide a plausible mechanism for
completing the catalytic act. Residue 35, glutamic acid (Glu-35), is about 3Å from the
-O- bridge that is to be broken. The free carboxyl group of glutamic acid is a
hydrogen ion donor and available to transfer H+ to the oxygen atom.
This would break the already-strained bond between the oxygen atom and the
carbon atom of ring 4.
Now having lost an electron, the carbon
atom acquires a positive charge. Ionized carbon is normally very unstable, but the
attraction of the negatively-charged carboxyl ion of Asp-52 could stabilize it long enough for an -OH ion (from a
spontaneously dissociated water molecule) to unite with the carbon. Even at pH
7, water spontaneously dissociates to produce H+ and OH-
ions. The hydrogen ion (H+) left over can replace that lost by Glu-35.
In either case, the
chain is broken, the two fragments separate from the enzyme, and the enzyme is
free to attach to a new location on the bacterial cell wall and continue its
work of digesting it.
Regulatory Enzymes
All enzymes exhibit
various features that could conceivably be elements in the regulation of their
activity in living cells. All have a characteristic optimum pH, which makes
possible alteration of their catalytic rates with changes in intracellular pH.
The rates of all enzymatic reactions also depend on the substrate
concentration, which may vary significantly under intercellular conditions.
Moreover, many enzymes require either metal ions, such as Mg2+ or K+,
or coenzymes for activity, suggesting that fluctuations in the concentration
of these metals or coenzymes in the cell can regulate enzyme activity. However,
over and above these properties of all enzymes, some enzymes possess other
properties that specifically endow them with regulatory roles in metabolism.
Such more highly specialized forms are called reguiatory enzymes. There are two major types of regulatory enzymes: (1)
allosteric enzymes, whose
catalytic activity is modulated through the noncovalent binding of a specific
metabolite at a site on the protein other than the catalytic site, and (2) covalently modulated enzymes, which are
interconverted between active and inactive forms by the action of other enzymes.
Some of the enzymes in the second class also respond to noncovalent allosteric
modulators. These two types of regulatory enzymes are responsive to alterations
in the metabolic state of a cell or tissue on a relatively short time scale —
allosteric enzymes within seconds and covalently regulated enzymes within
minutes.
Allosteric Enzymes
In many multienzyme
systems the end product of the reaction sequence may act as a specific
inhibitor of an enzyme at or near the beginning of the sequence, with the
result that the rate of the entire sequence of reactions is determined by the
steady-state concentration of the end product. The classical example is the
multienzyme sequence catalyzing the conversion of L-threonine to L-isoleucine,
which occurs in five enzyme-catalyzed steps. The first enzyme of the sequence,
L-threonine dehydratose, is strongly inhibited by L-isoleucine, the end
product, but not by any other intermediate in the sequence. The kinetic
characteristics of the inhibition by isoleucine are atypical; the inhibition is
neither competitive with the substrate L-threonine, nor is it noncompetitive.
Isoleucine is quite specific as an inhibitor; other amino acids or related
compounds do not inhibit. This type of inhibition is variously called end-product inhibition or feedback inhibition. The first enzyme in
this sequence, that which is inhibited by the end product, is called an allosteric enzyme. The term allosteric
denotes "another space" or "another structure"; allosteric enzymes possess, in
addition to the catalytic site, the "other space," to which the specific
effector 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
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.
Mechanism of the Regulatory Activity of Allosteric Enzymes
Much attention has been
focused on the molecular mechanisms by which the binding of the modulator to
the regulatory site of an allosteric enzyme can change the activity of the
catalytic site. One of the most fruitful avenues of research and speculation
has been evoked by the striking functional similarities between allosteric
enzymes and hemoglobin.
Two types of models have been
proposed for the cooperative interactions of the subunits of hemoglobin - the sequential model and the symmetry model. These models are also
applicable to allosteric enzymes having multiple subunits, which are assumed
to occur in two different conformations.
According to the symmetry model,
binding the first substrate molecule in creases the tendency of the remaining
subunits to undergo transition to the high-affinity form through an all-or-none
effect, all subunits being either in their low-affinity or their high-affinity
forms.
The sequential model also postulates
that the catalytic subunits have two conformational states, but it differs from
the symmetry model in postulating that the subunits may un-undergo individual
sequential changes in conformation; between the all-on and all-off states
there may be many intermediate conformational states of the enzyme molecule,
each having its own intrinsic catalytic activity.
Usage enzymes in medical practice
Enzymo-pathologies –
disoders of enzymes action. Enzymo-pathologies takes place actually at any disease
(it’s so called secondary enzymo-pathologies). But there also are more than one
thousand different forms of congenital molecular pathologies (primary
enzymo-pathologies). Mutations (genetic disorders) were found to be the causes
of these kind of enzymo-pathologies.
The
examples of primary enzymo-pathologies:
1)
Enzyme phenylalanine 4-monooxygenase
is absent in about
http://www.youtube.com/watch?v=hpaki7F4HR0
http://www.youtube.com/watch?v=CWfrVS4Bm1Y&feature=related
2)
The absence of homogentisic acid
1,2-dioxygenase causes urinary excretion of phenylpyruvic and homogentisic
acids. The urinary of people genetically defective in homogentisic acid
1,2-dioxygenase contains homogentisic acid, which when made alkaline and
exposed to oxygen, turns dark because it is oxidized and polimerized to a black
melanin pigment. This condition is known as alkaptonuria.
Patients with this condition have abnormal pigmentation of the connective
tissue.
3)
D-galactose is converted into D-glucose in the liver by special reactions which
have attracted much attention because they are subject to genetic defects in
man, resulting in different forms of the hereditary disease galactosemia. The deffect or absence of
enzyme galactose 1-phosphate
uridylyltransferase causes the increase of D-galactose in the blood.
Galactose 1-phosphate uridylyltransferase
is present in normal fetal liver but is lacking in infants with
galactosemia. Galactosemic infants suffer from cataract of the lens of the eye
as well as mental disorders. This condition can be successfully treated by
withholding milk and other sources of galactose from the diet during infancy
and childhood.
http://www.youtube.com/watch?v=lKaNgfya-DA
Enzymo-therapy.
In digestive tract diseases (deficit of digestive enzymes) pepsin and
HCI, enzymes of pancreas (pancreatin, oraza, panzinorm) are recomended to use.
Proteolytic enzymes trypsin and
chemotrypsin are used for processing of wounds in burns, ulcers for the a
proteolysis and deleting of necrotic tissues.
Trypsin,
ribonuclease, DNA-ase are used for the splitting and deleting of fibrin from
the pleural cavity, for the dilution and deleting of sputum from the
respiratory ways in acute and chronic respiratory diseases, for the
thrombophlebitis treatment.
Fibrinolysin, streptokinase are used for the splitting of thrombus.
Hyaluronidase (lidase) is used for the acceleration of different
medicines penetration into the biological tissues as well as for the resorption
of scars and hematoma.
Cytochrome C is used in the intoxication of CO, H2S and other
substances oppressing the tissue respiration.
Glucoso-oxidase is used for the lavage of wounds and burns as antiseptic.
Thrombin is used for preventing and stop of bleeding.
The using of coenzymes. ATP is
used in heart diseases, muscle dystrophia.
TPP
(thiamin pyrophosphate) is used in heart diseases, and nervous system
pathology.
FMN
(flavinmononucleotide) is used for treatment of skin diseases, keratitis,
conjunctivitis.
NAD
and NADP are used for improvement of oxidative-reduction processes in organism.
The
using of inhibitors of enzymes. Inhibitors of proteolytic enzymes (kontrical,
trasilol) are used in acute pancreatitis for inhibition trypsin, chemotrypsin
activity.
Diacarb
– inhibitor of carbohydrase. This enzyme regulates the metabolism of Na+
and K+ in kidney canaliculus and diuresis.
Fat-soluble vitamins
Although fat-soluble vitamins have
been studied intensively and widely used in human nutrition, we know less about
their specific biological function than about the water-soluble vitamins.
Vitamin A.
Vitamin A occurs in two common forms,
vitamin A1, or retinol, the form most common in
mammalian tissues and marine fishes, and vitamin, A2, common in
freshwater fishes. Both are isoprenoid compounds containing a six-membered
carbocyclic ring and an eleven-carbon side chain.
http://www.youtube.com/watch?v=dcw1m31zuTE
Vitamin A
Vitamin A
consists of three biologically active molecules, retinol, retinal
(retinaldehyde) and retinoic acid.
|
|
All-trans-retinal |
11-cis-retinal |
|
|
Retinol |
Retinoic Acid |
Carotenoids are provitamins of
vitamin A. Carotenoids widely distributed in plants, particularly a-, b-, and g-carotene. The carotenes have no vitamin A activity but are converted
into vitamin A by enzymatic reactions in the intestinal mucosa and the liver. b-Carotene, a symmetrical molecule, is cleaved in its center to yield
two molecules of retinol. Retinol occurs in the tissues of mammals and is
transported in the blood.
In vitamin A deficiency
young persons fail to grow, the bones and nervous system fail to develop
properly, the skin becomes dry and thickened, the kidneys and various glands
degenerate, and both males and females become sterile.
Although all
tissues appear to be disturbed by vitamin A deficiency, the eyes are most
conspicuously affected. In infants and young children the condition known as xerophthalmia ("dry eyes") is
an early symptom of deficiency and is a common cause of blindness in some
tropical areas where nutrition is generally poor. In adults an early sign of
vitamin A deficiency is nightblindness,
a deficiency in dark adaptation, which is often used as a diagnostic test.
Detailed
information is available on the role of vitamin A in the visual_cycle in vertebrates. The human retina contains two types of
light-sensitive photoreceptor cells. Rod-cells
are adapted to sensing low light intensities, but not colors; they are the
cells involved in night vision, whose function is impaired by vitamin A
deficiency. Cone cells, which sense
colors, are adapted for high light intensities.
Retinal rod cells contain many membrane
vesicles that serve as light receptors. About one-half of the protein in the
membrane of these vesicles consists of the light-absorbing protein rhodopsin (visual purple). Rhodopsin consists of a protein, opsin, and tightly bound 11-cis-retinal, the aldehyde of vitamin
A. When rhodopsin is exposed to light, the bound 11-cis-retinal undergoes transformation
into all-trans-retinal, which causes a substantial change in the configuration
of the retinal molecule. This reaction is nonenzymatic. The isomerization of
retinal is followed by a series of other molecular changes, ending in the
dissociation of the rhodopsin to yield free opsin and all-trans-retinal, which
functions as a trigger setting off the nerve impulse.
11-cis-retinal all-trans-retinal
In order for rhodopsin to be
regenerated from opsin and all-trans-retinal, the latter must undergo
isomerization back to 11-cis-retinal. This appears to occur in a sequence of enzymatic
reactions catalyzed by two enzymes:
retinal-reductase
all-trans-retinal
+ NADH + H+ → all-trans-retinol + NAD+
retinol-isomerase
all-trans-retinol →
11-cis-retinol
retinal-reductase
11-cis-retinol
+ NAD+ → 11-cis-retinal + NADH + H+
The 11-cis-retinal so formed now
recombines with opsin to yield rhodopsin, thus completing the visual cycle.
Since vitamin A deficiency affects
all tissues of mammals, not the retina alone, the role of retinal in the visual
cycle does not represent the entire action of vitamin A. It appears possible
that vitamin A may play a general role in:
- the transport of Ca2+
across certain membranes; such a more general role might explain the effects of
vitamin A deficiency and excess on bony and connective tisues;
-
processes of growth and
cell differentiation;
-
processes of
glycoproteins formation whoch are the components of the biological mucosa .
The vitamin A requirement of man -
1,5-2 milligram per day.
Vitamin A is met in large part by
green and yellow vegetables, such as lettuce, spinach, sweet potatoes, and
carrots, which are rich in carotenes. Fish-liver oils are particularly rich in
vitamin A. However, excessive intake of vitamin A is toxic and leads to easily
fractured, fragile bones in children, as well as abnormal development of the
fetus.
Vitamin D
Most important are vitamin D2,
or ergocalciferol, and vitamin
D3, or cholecalciferol, the form normally found
in mammals. These compounds may be regarded as steroids.
It is now known that 7-dehydrocholesterol in the skin is the
natural precursor of cholecalciferol in man; the conversion requires
irradiation of the skin by sunlight. On a normal unsupplemented diet this is
the major route by which people usually acquire vitamin D.
Vitamin D is a steroid hormone that functions to regulate specific gene
expression following interaction with its intracellular receptor. The
biologically active form of the hormone is 1,25-dihydroxy vitamin D3
(1,25-(OH)2D3, also termed calcitriol). Calcitriol
functions primarily to regulate calcium and phosphorous homeostasis.
http://www.youtube.com/watch?v=JwPVibQ6_3Y&feature=related
http://www.youtube.com/watch?v=onSPZ0aBUKM&feature=related
http://www.youtube.com/watch?v=xwNhd2pQL0k&feature=related
|
|
Ergosterol |
Vitamin D2 |
|
|
7-Dehydrocholesterol |
Vitamin D3 |
Cholecalciferol is converted into its
derivative - 25-hydroxycholecalciferol. This product is more active
biologically than cholecalciferol and it has been found to be the main
circulating form of vitamin D in animals, formed in the liver. But
25-hydroxycholecalciferol was found to be metabolized further to
1,25-dihydroxycholecalciferol in kidneys. This compound is still more active;
its administration produces rapid stimulation of Ca2+ absorption by
the intestine.
|
|
25-hydroxyvitamin D3 |
1,25-dihydroxyvitamin D3 |
So the kidney is the site of formation of
1,25-dihydroxycholecalciferol, which now appears to be the biologically active
form of vitamin D, capable of acting directly on its major targets, the small
intestine and the bones.
1,25-dihydroxycholecalciferol
promotes absorption of Ca2+ from the intestine into the blood,
through its ability to stimulate the biosynthesis of specific protein(s) that
participate in transport or binding of Ca2+ in the intestinal
mucosa. This role of 1,25-dihydroxycholecalciferol is integrated with the
action of parathyroid hormone. Whenever the Ca2+
concentration of the blood becomes lower than normal, the parathyroid glands
secrete larger amounts of parathyroid hormone. This hormone acts on the kidney,
stimulating it to produce more 1,25-dihydroxycholecalciferol from its
precursor 25-hydroxycholecalciferol.
Rickets, a disease of growing bone,
is developed in the deficiency of vitamin D in organism.
http://www.youtube.com/watch?v=n7vybcT9_F4
As with vitamin A, excessive intake of vitamin D
causes the bones to become fragile and to undergo multiple fractures,
suggesting that both vitamins play a role in biological transport and
deposition of calcium.
Most natural foods contain little of vitamin D;
vitamin D in the diet comes largely from fish-liver oils, liver, yoke of eggs,
butter. Vitamin D preparations available commercially are products of the
ultraviolet irradiation of ergosterol from yeast.
About 2,5-10 mkg of vitamin D is required by an adult
daily and 12-25 mkg by children. The vitamin can be stored in sufficient
amounts in the liver for a single dose to suffice for some weeks.
|
a-Tocopherol |
Vitamin E was first
recognized as a factor in vegetable oils that restores fertility in rats grown
on cow's milk alone and otherwise incapable of bearing young. It was isolated
from wheat germ and was given the name tocopherol. Several different
tocopherols having vitamin E activity have been found in plants; the most
active and abundant is a-tocopherol.
The deficiency of
tocopherol produces many other symptoms besides infertility in male and female,
e.g., degeneration of the kidneys, the deposition of brown pigments in lipid
depots, necrosis of the liver, and dystrophy, or wasting, of skeletal muscles.
Tocopherols have been found to have
antioxidant activity; i.e., they prevent the autoxidation of highly unsaturated
fatty acids when they are exposed to molecular oxygen. One of the functions of
tocopherol may be to protect highly unsaturated fatty acids in the lipids of
biological membranes against the deleterious effects of molecular oxygen.
Normally, autoxidation products of unsaturated fats do not occur in the
tissues, but in tocopherol deficiency they are detectable in the fat depots,
liver, and other organs.
Due to the hydrophobic side radical
tocopherol can be built into the phospholipid matrix of biological membranes
and stabilize the mobility and microviscosity of membrane proteins and lipids.
Tocopherol is the most potent natural antioxidant.
About 10-20 mg of vitamin E is required per day.
The most abundant sources of vitamin E are oils (sunflower, corn, soybean oils), fresh vegetables,
animal stuffs (meat, butter, egg yoke).
http://www.youtube.com/watch?v=8oRUF_g-J3k&feature=related
Vitamin K
The K vitamins exist naturally as K1
(phylloquinone) in green vegetables and K2 (menaquinone) produced by
intestinal bacteria and K3 is synthetic menadione. When
administered, vitamin K3 is alkylated to one of the vitamin K2
forms of menaquinone.
|
|
|
Vitamin K1 |
Vitamin K2 |
Vitamin |
Vitamin K was first discovered as a nutritional factor required
for normal blood-clotting time. At least two forms of vitamin K are known; vitamin K2 is believed to be the
active form. Vitamin K deficiency cannot readily be produced in rats and other
mammals because the vitamin is synthesized by intestinal bacteria.
http://www.youtube.com/watch?v=DVGsnlVCoeA
http://www.youtube.com/watch?v=WI24c2LYFug&feature=related
The only known result of vitamin K
deficiency is a failure in the biosynthesis of the enzyme proconvertin in the liver. This enzyme catalyzes a step in a
complex sequence of reactions involved in the formation of prothrombin, the precursor of thrombin, a protein that accelerates the
conversion of fibrinogen into fibrin, the insoluble protein
constituting the fibrous portion of blood clots.
The compound dicumarol, an analog of vitamin K,
produces symptoms in animals resembling vitamin K deficiency; it is believed to
block the action of vitamin K. Dicumarol is used in clinical medicine to
prevent clotting in blood vessels. Dicumarol is the antivitamin of vitamin K.
Some evidence indicates that vitamin
K may function as a coenzyme in a specialized route of electron transport in
animal tissues; since vitamin K is a quinone which can be reduced reversibly to
a quinol, it may serve as an electron carrier.
Function
Food Source
Effective With
Increased
Intakes Needed
Used
For
Destroyed By
Symptoms
of Deficiency
In
babies:
Deficiency
Caused By
In
Babies:
In
Adults:
Deficiency
Leads To
Hypovitaminos of vitamin K in man can
be developed in liver diseases when there is the decrease of bile acids amount
in intestine and as result the inhibition of fat soluble substances absorption
is observed.
Vitamin K is produced by many microorganisms in the
intestine. also Plants (cabbage, tomato, lettuce)are natural sources of vitamin
K.
Adult person requires 200-300 mkg of vitamin K per
day.
References:
1.
John Mc Murry, Mary E. Castellion.
General, Organic and Biological Chemistry.- New Jersy: Prentice Hall, 1992.-
764 p.
2.
John W. Suttie.
Introduction to Biochemistry. –
3.
Robert K. Murray, Daryl
K. Granner. Harper’s illustrated Biochemistry. –
4.
VK Malhotra. Biochemistry
for students. –
5.
Lehninger A. Principles
of Biochemistry. –
6.
Stryer L. Biochemistry. –
Investigation of water soluble (coenzyme) vitamins functional role in metabolism and
cell functions realization.
Vitamins are nutrients
required in tiny amounts for essential metabolic
reactions in the body. The term vitamin does not include other
essential nutrients such as dietary minerals,
essential fatty acids, or essential amino acids, nor does it encompass the
large number of other nutrients that promote health but that are not essential
for life.
Vitamins
are bio-molecules
that act both as catalysts and substrates in chemical
reactions. When acting as a catalyst, vitamins are bound to enzymes and are called cofactors.
(For example, vitamin K
forms part of the proteases involved in blood clotting.)
Vitamins also act as coenzymes to carry chemical groups between enzymes.
(For example, folic acid carries various forms of carbon
groups–methyl, formyl or methylene–in the cell.)/
Until the 1900s, vitamins were obtained solely through
food intake. Many food sources contain different ratios of vitamins. Therefore,
if the only source of vitamins is food, changes in diet will alter the types
and amounts of vitamins ingested. However, as many vitamins can be stored by
the body, short-term deficiencies (which, for example, could occur during a
particular growing season) do not usually cause disease.
Vitamins have been produced
as commodity chemicals and made widely available as inexpensive pills for
several decades,[2]
allowing supplementation of the dietary intake.
Difference
from water soluble vitamins: water
soluble vitamins are included into coenzymes, don't have provitamins, are not
included into the membranes, and hypervitaminoses
are not peculiar for them.
With exception of
vitamin B6 and B12, they are readily excreted in urine
without appreciable storage, so frequent consumption becomes necessary. They
are generally nontoxic when present in excess of needs, although symptoms may be
reported in people taking megadoses of niacin,
vitamin C, or pyridoxine (vitamin B6). All the B vitamins function
as coenzymes or cofactors, assisting in the activity of important enzymes and
allowing energy-producing reactions to proceed normally. As a result, any lack of water-soluble vitamins mostly affects growing
or rapidly metabolizing tissues such as skin, blood, the digestive tract, and
the nervous system. Water-soluble
vitamins are easily lost with overcooking.
Water-soluble vitamins and their
characteristics. |
|
|
||||||
Common food sources |
Major functions |
Deficiency symptoms |
Overconsumption
symptoms |
Stability in foods |
|
|
||
Vitamin C (abscorbic acid) |
|
|
||||||
Citrus fruits, broccoli,
strawberries, melon, green pepper, tomatoes, dark green vegetables, potatoes. |
Formation of collagen (a component
of tissues), helps hold them together; wound healing; maintaining blood
vessels, bones, teeth; absorption of iron, calcium, folacin; production of
brain hormones, immune factors; antioxidant. |
Bleeding gums; wounds don't heal;
bruise easily; dry, rough skin; scurvy; sore joints and bones; increased
infections. |
Nontoxic under normal conditions;
rebound scurvy when high doses discontinued; diarrhea, bloating, cramps;
increased incidence of kidney stones. |
Most unstable under heat, drying,
storage; very soluble in water, leaches out of some vegetables during
cooking; alkalinity (baking soda) destroys vitamin C. |
|
|
||
Thiamin (vitamin B1 ) |
|
|
||||||
Pork, liver, whole grains, enriched
grain products, peas, meat, legumes. |
Helps release energy from foods;
promotes normal appetite; important in function of nervous system. |
Mental confusion; muscle weakness,
wasting; edema; impaired growth; beriberi. |
None known. |
Losses depend on cooking method,
length, alkalinity of cooking medium; destroyed by sulfite used to treat
dried fruits such as apricots; dissolves in cooking water. |
|
|
||
Riboflavin (vitamin B2) |
|
|
||||||
Liver, milk, dark green vegetables,
whole and enriched grain products, eggs. |
Helps release energy from foods;
promotes good vision, healthy skin. |
Cracks at corners of mouth;
dermatitis around nose and lips; eyes sensitive to light. |
None known. |
Sensitive to light; unstable in
alkaline solutions. |
|
|||
Niacin (nicotinamide, nicotinic acid) |
|
|
||||||
Liver, fish, poultry, meat,
peanuts, whole and enriched grain products. |
Energy production from foods; aids
digestion, promotes normal appetite; promotes healthy skin, nerves. |
Skin disorders; diarrhea; weakness;
mental confusion; irritability. |
Abnormal liver function; cramps;
nausea; irritability. |
|
|
|||
Vitamin B6 (pyridoxine,
pyridoxal, pyridoxamine) |
|
|
||||||
Pork, meats, whole grains and
cereals, legumes, green, leafy vegetables. |
Aids in protein metabolism,
absorption; aids in red blood cell formation; helps body use fats. |
Skin disorders, dermatitis, cracks
at corners of mouth; irritability; anemia; kidney stones; nausea; smooth
tongue. |
None known. |
Considerable losses during cooking. |
|
|||
|
|
|
|
|
|
|
|
|
Folacin (folic acid) |
|
||||
Liver, kidney, dark green leafy
vegetables, meats, fish, whole grains, fortified grains and cereals, legumes,
citrus fruits. |
Aids in protein metabolism;
promotes red blood cell formation; prevents birth defects of spine, brain;
lowers homocystein levels and thus coronary heart disease risk. |
Anemia; smooth tongue; diarrhea. |
May mask vitamin B12
deficiency (pernicious anemia). |
Easily destroyed by storing,
cooking and other processing. |
|
Vitamin B12 |
|
||||
Found only in animal foods: meats,
liver, kidney, fish, eggs, milk and milk products, oysters, shellfish. |
Aids in building of genetic
material; aids in development of normal red blood cells; maintenance of
nervous system. |
Pernicious anemia, anemia;
neurological disorders; degeneration of peripheral nerves that may cause
numbness, tingling in fingers and toes. |
None known. |
|
|
Pantothenic acid |
|
||||
Liver, kidney, meats, egg yolk,
whole grains, legumes; also made by intestinal bacteria. |
Involved in energy production; aids
in formation of hormones. |
Uncommon due to availability in
most foods; fatigue; nausea, abdominal cramps; difficulty sleeping. |
None known. |
About half of pantothenic acid is lost
in the milling of grains and heavily refined foods. |
|
Thiamin
(Vitamin B1)
Thiamine
or thiamin, also known as vitamin
B1, is a colorless compound with chemical formula C12H17N4OS.
It is soluble in water and insoluble in alcohol.
Thiamine decomposes if heated. Its
chemical structure contains a pyrimidine
ring and a thiazole ring.Thiamine was first
discovered in 1910 by Umetaro Suzuki
in Japan when researching how rice bran cured patients of Beriberi.
He named it aberic acid. Thiamine diphosphate (ThDP) or thiamine
pyrophosphate (TPP) is a coenzyme
for pyruvate dehydrogenase,
α-ketoglutarate dehydrogenase, branched-chain
Thiamin
pyrophosphate
alpha-keto acid dehydrogenase, and transketolase. The first two of these enzymes function in the metabolism of carbohydrates, while transketolase
functions in the pentose phosphate pathway to synthesize NADPH and the pentose sugars deoxyribose and ribose. In general, TPP
functions as a cofactor for enzymes that catalyze the dehydrogenation
(decarboxylation and subsequent conjugation to Coenzyme A) of alpha-keto acids.
TPP is synthesized by the enzyme thiamine
pyrophosphokinase,
which requires free thiamine, magnesium,
and adenosine triphosphate.
Good sources: Thiamine is found naturally in the following foods, each of which
contains at least 0.1mg of the vitamin per 28-100g (1-3.5oz): Green peas, Spinach, Liver, Beef, Pork, Navy beans, Nuts, Pinto beans, Soybeans, Whole-grain and Enriched
Cereals, Breads, Yeast, and Legumes.
http://www.youtube.com/watch?v=Q5BCmsixuqM
Thiamin functions as the coenzyme thiamin pyrophosphate (TPP) in the metabolism of carbohydrate and in conduction of nerve impulses. Thiamin
deficiency causes beri-beri, which is frequently seen in parts of the world
where polished (white) rice or unenriched white flour are predominantly eaten.
http://www.youtube.com/watch?v=PD_CoEngu4M&feature=related
There are three basic expressions of
beriberi: childhood, wet, and dry. Childhood beriberi stunts growth in infants
and children. Wet beriberi is the classic form, with swelling due to fluid
retention (edema) in the lower limbs
that spreads to the upper body, affecting the heart and leading to heart
failure. Dry beriberi affects peripheral nerves, initially causing tingling or
burning sensations in the lower limbs and progressing to nerve degeneration,
muscle wasting, and weight loss.
Thiamine-deficiency disease in North America commonly occurs in people with
heavy alcohol consumption and is called Wernicke-Korsakoff syndrome. It is
caused by poor food intake and by decreased absorption and increased excretion caused by alcohol consumption.
Riboflavin (Vitamin B2)
Riboflavin is stable when heated in
ordinary cooking, unless the food is exposed to ultraviolet radiation
(sunlight). To prevent riboflavin breakdown, riboflavin-rich foods such as
milk, milk products, and cereals are packaged in opaque containers. Riboflavin
is a component of two coenzymes—flavin mononucleotide (FMN) and flavin adenine
dinucleotide (FAD)—that act as hydrogen carriers when carbohydrates and fats
are used to produce energy. It is helpful in maintaining good vision and
healthy hair, skin and nails, and it is necessary for normal cell growth.
Thiamin
pyrophosphate
Riboflavin deficiency
causes a condition known as ariboflavinosis, which is marked by cheilosis
(cracks at the corners of the mouth), oily scaling of the skin, and a red, sore
tongue. In addition, cataracts may
occur more frequently with riboflavin deficiency. A deficiency of this nutrient is usually a part of
multinutrient deficiency and does not occur in isolation. In North America, it
is mostly observed in alcoholics, elderly persons with low income or depression, and people with poor eating
habits, particularly those who consume highly refined and
fast foods and those who do not consume milk and milk products.
http://www.youtube.com/watch?v=qpvNaGIJMzw
Unlike fat-soluble vitamins, water-soluble
vitamins are easily lost during cooking and processing. The body does not store
excess quantities of most water-soluble vitamins, so foods bearing them must be
consumed frequently.
Niacin
(Vitamin B5)
|
|
Nicotinamide |
Nicotinic Acid |
Niacin exists in two
forms, nicotinic acid and nicotinamide. Both forms are readily absorbed from
the stomach and the small intestine. Niacin is stored in small amounts in the
liver and transported to tissues, where it is converted to coenzyme forms. Any
excess is excreted in urine. Niacin is one of the most stable of the B
vitamins. It is resistant to heat and light, and to both acid and alkali
environments. The human body is capable of converting the amino acid tryptophan to niacin when needed. However, when both
tryptophan and niacin are deficient, tryptophan is used for protein synthesis.
Structure
of NAD+
There are two coenzyme
forms of niacin: nicotinamide adenine dinucleotide (NAD+) and
nicotinamide adenine dinucleotide phophate (NADP+). They both help
break down and utilize proteins, fats, and carbohydrates for energy. Niacin is
essential for growth and is involved in hormone
synthesis.
Pellagra results from a
combined deficiency of niacin and tryptophan. Long-term deficiency leads to
central nervous system dysfunction manifested as confusion, apathy,
disorientation, and eventually coma and death. Pellagra
is rarely seen in industrialized countries, where it may be observed in people
with rare disorder of tryptophan metabolism (Hartnup's disease), alcoholics,
and those with diseases that affect food intake.
http://www.youtube.com/watch?v=UrDeVyiXzyg&feature=related
The liver can synthesize niacin from the essential aminoacid tryptophan, but
the synthesis is extremely slow; 60 mg of tryptophan are required to make one
milligram of niacin. Dietary niacin deficiency tends to occur only in areas
where people eat corn, the only grain low in niacin, as a staple food, and
that don't use lime during maize (corn) meal/flour production. Alkali lime
releases the tryptophan from the corn so that it can be absorbed in the gut,
and converted to niacin.
|
Niacin plays an important role in the
production of several sex and stress-related hormones, particularly those made
by the adrenal gland. Niacin, when taken in large doses, increases the level of
high density lipoprotein (HDL) or "good" cholesterol in blood, and is sometimes
prescribed for patients with low HDL, and at high risk of heart attack. Niacin
(but not niacinamide) is also used in the treatment of hyperlipidemia because it reduces very low density lipoprotein
(VLDL), a precursor of low density lipoprotein
(LDL) or "bad" cholesterol, secretion from the liver, and inhibits cholesterol synthesis.
The main problem with the clinical use of
niacin for dyslipidemia is the occurrence of skin flushing, even with moderate
doses.
Recommended intake is expressed as
milligrams of niacin equivalents (NE) to account for niacin synthesized from
tryptophan. High doses taken orally as nicotinic acid at 1.5 to
The nicotinamide form
of niacin in multivitamin and B-complex tablets do not work for this purpose.
Supplementation should be under a physician's guidance.
http://www.youtube.com/watch?v=MLFZ8CsrJqU&feature=related
Pantothenic
Acid (Vitamin B3)
|
Pantothenic Acid |
Pantothenic acid, also called vitamin
B3,
is a water-soluble vitamin required to
sustain life. Pantothenic acid is needed to form coenzyme-A (CoA), and is critical
in the metabolism and synthesis of carbohydrates, proteins, and fats. Its name
is
derived from the Greek pantothen meaning "from everywhere" and small quantities of
pantothenic acid are found in nearly every food, with high amounts in
whole grain cereals,
legumes, eggs, meat, and royal jelly
Pantothenic acid is stable in moist heat.
It is destroyed by vinegar (acid), baking soda (alkali), and dry heat.
Significant losses occur during the processing and refining of foods.
Pantothenic acid is released from coenzyme A in food in the small intestine.
After absorption, it is transported to tissues, where coenzyme A is
resynthesized. Coenzyme A is essential for the formation of energy as adenosine
triphosphate (ATP) from carbohydrate, protein, alcohol, and fat.
Coenzyme A is also important in
the synthesis of fatty acids,
cholesterol, steroids, and the neurotransmitter acetylcholine, which
is essential for transmission of nerve impulses to muscles.
|
Coenzyme A |
Dietary deficiency occurs in conjunction
with other B-vitamin deficiencies. Pantothenic acid is used in the synthesis
of coenzyme A (abbreviated as CoA).
Coenzyme A may act as an acyl group carrier to form acetyl-CoA and other related
compounds; this is a way to transport carbon
atoms within the cell. The transfer of carbon atoms by coenzyme A is important
in cellular respiration, as well as the
biosynthesis of many important compounds such as fatty acids, cholesterol, and acetylcholine. Dietary deficiency occurs
in conjunction with other B-vitamin deficiencies. In studies, experimentally
induced deficiency in humans has resulted in headache, fatigue, impaired
muscle coordination, abdominal cramps, and vomiting.
In studies, experimentally
induced deficiency in humans has resulted in headache, fatigue, impaired muscle coordination, abdominal cramps, and
vomiting.
Biotin
(Vitamin B8)
|
Biotin
is a water soluble vitamin and a member of Vitamin B complex. Also known
as Vitamin H, Bios II, Co-enzyme R. Its natural form is D-biotin.
It was isolated from liver in 1941 by Dr. Paul Gyorgy.
http://www.youtube.com/watch?v=o9lJoKoF4DE
FUNCTION
FOOD
SOURCE
EFFECTIVE WITH
INCREASED INTAKES
NEEDED
USED
FOR
DESTROYED
BY
SYMPTOMS
OF DEFICIENCY
In
babies:
In
adults:
DEFICIENCY
LEADS TO
SYMPTOMS
OF TOXICITY
High quality
Vitamin B (Biotin) can be purchased from Global Herbal Supplies
Biotin is the most stable of B vitamins.
It is commonly found in two forms: the free vitamin and the protein-bound
coenzyme form called biocytin. Biotin is absorbed in the small intestine, and
it requires digestion by enzyme biotinidase, which is present in the small
intestine. Biotin is synthesized by bacteria
in the large intestine, but its absorption is questionable. Biotincontaining
coenzymes participate in key reactions that produce energy from carbohydrate
and synthesize fatty acids and protein.
Avidin is a protein in raw egg white, which can bind to the biotin in
the stomach and decrease its absorption. Therefore, consumption of raw whites
is of concern due to the risk of becoming biotin deficient. Cooking the egg
white, however, destroys avidin. Deficiency may develop in infants born with a genetic defect that results in reduced
levels of biotinidase. In the past, biotin deficiency was observed in infants
fed biotin-deficient formula, so it is now added to infant formulas and other
baby foods.
Vitamin B6
|
|
|
|
Pyridoxal |
|
http://www.youtube.com/watch?v=zwUc5gHoF_U&feature=related
Pyridoxal, pyridoxamine and pyridoxine are
collectively known as vitamin B6. All three compounds are
efficiently converted to the biologically active form of vitamin B6,
pyridoxal phosphate. This conversion is catalyzed by the ATP requiring enzyme,
pyridoxal kinase.
|
Pyridoxal Phosphate |
http://www.youtube.com/watch?v=Q9yBs6wvMFc
Vitamin B6 is present in three
forms: pyridoxal, pyridoxine, and pyridoxamine. All forms can be converted to
the active vitamin-B6 coenzyme in the body. Pyridoxal phosphate
(PLP) is the predominant biologically active form. Vitamin B6 is not
stable in heat or in alkaline conditions, so cooking and food processing reduce
its content in food. Both coenzyme and free forms are absorbed in the small
intestine and transported to the liver, where they are phosphorylated and released
into circulation, bound to albumin for transport to tissues. Vitamin B6
is stored in the muscle and only excreted in urine when intake is excessive.
PLP participates in amino acid synthesis
and the interconversion of some amino acids. It catalyzes a step in the synthesis of hemoglobin, which is needed to transport oxygen in blood. PLP helps maintain blood glucose levels by facilitating the release of glucose from liver
and muscle glycogen. It also plays a
role in the synthesis of many neurotransmitters important for brain function.
This has led some physicians to prescribe megadoses of B6 to
patients with psychological problems
such as depression and mood swings, and to some women for premenstrual syndrome
(PMS). It is unclear, however, whether this therapy is effective. PLP
participates in the conversion of the amino acid tryptophan to niacin and helps
avoid niacin deficiency. Pyridoxine affects immune function, as it is essential
for the formation of a type of white blood cell.
Populations at risk of vitamin-B6 deficiency include
alcoholics and elderly persons who consume an inadequate diet. Individuals
taking medication to treat Parkinson's disease or tuberculosis may take extra vitamin B6 with physician
supervision. Carpal tunnel syndrome, a nerve disorder of the wrist, has also
been treated with large daily doses of B6. However, data on its
effectiveness are conflicting.
Folic
Acid, Folate, Folacin (Vitamin B9)
|
Folacin or folate, as it is usually
called, is the form of vitamin B9 naturally present in foods,
whereas folic acid is the synthetic form added to fortified foods and supplements. Both forms are absorbed in the
small intestine and stored in the liver. The folic acid form, however, is more
efficiently absorbed and available to the body. When consumed in excess of
needs, both forms are excreted in urine and easily destroyed by heat,
oxidation, and light.
Folic acid is a water soluble
vitamin and is a member of the Vitamin B complex. Also known as Folacin,
pteroyl-L-glutamic acid (PGA), vitamin Bc or vitamin M. Folic acid and its
derivatives (mostly the tri and heptaglutamyl peptides) are widespread in
nature. It is a specific growth factor for certain micro-organisms. Found
in yeast and liver in 1935.
All forms of this vitamin
are readily converted to the coenzyme form called tetrahydrofolate (THFA),
which plays a key role in transferring single-carbon methyl units during the
synthesis of DNA and RNA, and in interconversions of amino
acids. Folate also plays an important role in the synthesis of
neurotransmitters. Meeting folate needs can improve mood and mental functions.
Function
Food Source
Effective With
Increased Intakes
Needed
Used For
Destroyed
By
Symptoms
of Deficiency
Deficiency
Leads To
Various
conditions relating to childbirth:
As
well as:
Symptoms
of Toxicity
Folic
Acid has a low toxicity but occasionally the following symptoms occur:
Long
term high doses may cause Vitamin B12 losses from the body
http://www.youtube.com/watch?v=4-pMZRxyasU&feature=related
http://www.youtube.com/watch?v=TLDodF9kkRo&feature=related
http://www.youtube.com/watch?v=_QFl7BnWhpQ&feature=related
Folate deficiency is one of the most
common vitamin deficiencies. Early symptoms are nonspecific and include
tiredness, irritability, and loss of appetite. Severe folate deficiency leads
to macrocytic anemia, a condition in
which cells in the bone marrow
cannot divide normally and red blood cells remain in a large immature form
called macrocytes. Large immature
cells also appear along the length of the gastrointestinal
tract, resulting in abdominal pain and diarrhea.
Pregnancy is a time of rapid cell
multiplication and DNA synthesis, which increases the need for folate. Folate
deficiency may lead to neural tube
defects such as spina bifida (failure of the spine to close properly during the
first month of pregnancy) and anencephaly (closure of the neural tube during
fetal development, resulting in part
of the cranium not being formed). Seventy percent of these defects could be
avoided by adequate folate status before conception, and it is recommended that
all women of childbearing age consume at least 400 micrograms (μg) of folic acid each day from fortified foods
and supplements. Other groups at risk of deficiency
include elderly persons and persons suffering from alcohol abuse or taking
certain prescription drugs.
Vitamin
B12
Vitamin B12 is found in its
free-vitamin form, called cyanocobalamin, and in two active coenzyme forms.
Absorption of vitamin B12 requires the presence of intrinsic factor,
a protein synthesized by acid-producing cells
of the stomach. The vitamin is absorbed in the terminal portion of the small
intestine called the ileum. Most of body's supply of vitamin B12 is
stored in the liver.
Vitamin B12
|
Cyanocobalamin |
Vitamin B12 is defficiently
conserved in the body, since most of it is secreted into bile and reabsorbed. This explains the slow development (about two years)
of deficiency in people with reduced intake or absorption. Vitamin B12
is stable when heated and slowly loses its activity when exposed to light,
oxygen, and acid or alkaline environments.
Vitamin B12 coenzymes help
recycle folate coenzymes involved in the synthesis of DNA and RNA, and in the
normal formation of red blood cells. Vitamin B12 prevents
degeneration of the myelin sheaths that cover nerves and help maintain normal
electrical conductivity through the nerves.
|
Active center of
tetrahydrofolate (THF). Note that the N5 position is the site of
attachment of methyl groups, the N10 the site for attachment of
formyl and formimino groups and that both N5 and N10
bridge the methylene and methenyl groups |
Vitamin-B12 deficiency results
in pernicious anemia, which is caused
by a genetic problem in the production of intrinsic factor. When this occurs,
folate function is impaired, leading to macrocytic anemia due to interference
in normal DNA synthesis. Unlike folate deficiency, the anemia caused by
vitamin-B12 deficiency is accompanied by symptoms of nerve
degeneration, which if left untreated can result in paralysis and death.
http://www.youtube.com/watch?v=IQ0Aet8FVVU&feature=related
http://www.youtube.com/watch?v=DQ7IHIgw1ic&feature=related
Since vitamin B12 is well conserved in the body, it is
difficult to become deficient from dietary factors alone, unless a person is a
strict vegan and consumes a
diet devoid of eggs and dairy for several
years. Deficiency is usually observed when B12
absorption is hampered by disease or surgery to the stomach or ileum, damage to
gastric mucosa by alcoholism, or
prolonged use of anti-ulcer medications that affect secretion of intrinsic
factor. Agerelated decrease in stomach-acid production also reduces absorption
of B12 in elderly persons. These groups are advised to consume
fortified foods or take a supplemental form of vitamin B12.
Choline
For many years, choline was not considered
a vitamin because the body makes enough of it to meet its needs in most age
groups. However, research now shows that choline production in the body is not
enough to cover requirements. Choline is not considered a B vitamin because it
does not have a coenzyme function and the amount in the body is much greater
than other B vitamins. Choline not only helps maintain the structural integrity
of membranes surrounding every cell in the body, but also can play a role in
nerve signaling, cholesterol transport, and energy metabolism. An
"adequate intake" is 550 milligrams per day for men and 425 milligrams
per day for women. Choline is widely found in foods, so it is unlikely that a
dietary deficiency will occur.
Vitamin
C (Ascorbic Acid)
|
In 1746, James Lind, a British physician,
conducted the first nutrition
experiment on human beings in an effort to find a cure for scurvy.
James
Lind (1716 – 1794),a British Royal Navy surgeon who, in 1774,
identified that a quality in fruit prevented the disease of scurvy in what was
the first recorded controlled experiment
However, it was not until nearly 200 years
later that ascorbic acid, or vitamin C, was discovered. Vitamin C participates
in many reactions by donating electrons as hydrogen atoms. In a reducing reaction, the electron in the hydrogen atom
donated by vitamin C combines with other participating molecules, making vitamin C a reducing agent, essential to the
activity of many enzymes. By neutralizing free
radicals, vitamin C may reduce the risk of heart disease, certain forms of cancer, and cataracts.
Vitamin C is needed to form and maintain
collagen, a fibrous protein that gives strength to connective tissues in skin,
cartilage, bones, teeth, and joints. Collagen is also needed for the healing of
wounds.
When added to meals, vitamin C increases
intestinal absorption of iron from
plant-based foods. High concentration of vitamin C in white blood cells enables the immune
system to function properly by providing protection against oxidative damage from free radicals
generated during their action against bacterial, viral, or fungal infections.
Vitamin C also recycles oxidized vitamin E for
reuse in cells, and it helps folic acid convert to its active form, (THF).
Vitamin C helps synthesize carnitine, adrenaline, epinephrine, the neurotransmitter serotonin, the thyroid hormone thyroxine, bile acids, and steroid hormones.
A deficiency of
vitamin C causes widespread connective tissue changes throughout the body.
Deficiencies may occur in people who eat few fruits and vegetables, follow
restrictive diets, or abuse alcohol and drugs. Smokers also have lower
vitamin-C status. Supplementation may be prescribed by physicians to speed the
healing of bedsores, skin ulcers,
fractures, burns, and after surgery. Research has shown that doses up to
Ascorbic
acid
Ascorbic acid is required in the diet of
only a few vertebrates — man, monkeys, the guinea pig, and certain fishes. Some
insects and other invertebrates also require ascorbic acid, but most other
higher animals and plants can synthesize ascorbic acid from glucose or other
simple precursors. Ascorbic acid is not present in microorganisms, nor does it
seem to be required.
Ascorbic acid is a
strong reducing agent, readily losing hydrogen atoms to become dehydroascorbic acid, which also has
vitamin C activity. However, vitamin activity is lost when the lactone ring of
dehydroascorbic acid is hydrolyzed to yield diketogulonic acid.
Ascorbic Dehydroascorbic Diketogulonic
acid
acid acid
Biological role of ascorbic acid:
-
acts as a cofactor in the
enzymatic hydroxylation of proline to hydroxyproline and in other
hydroxylation reactions;
-
inhibits the oxidation of
hemoglobin;
-
accelerates the oxidation
of glucose in pentose phosphate pathway;
-
reduces the disulfide
bonds to sulfhydryl bonds;
-
is necessary for
hydroxylation of cholesterol;
-
takes part in metabolism
of adrenaline;
-
is necessary for the
metabolism of mineral elements (Fe, Ca);
- accelerates the synthesis of
glycogen in liver.
While at sea in May 1747, Lind provided some
crewmembers with two oranges
and one lemon
per day, in addition to normal rations, while others continued on cider,
vinegar
or seawater, along with their normal rations. In the history of science this is considered to be the
first example of a controlled experiment comparing results on two populations
of a factor applied to one group only with all other factors the same.
In the hypovitaminosis of vitamin C
the disease scurvy is developed. Main clinical symptoms of scurvy: delicacy,
vertigo, palpitation, tachycardia, pain
in the area of heart, dyspnea, petechias, odontorrhagia, dedentition.
Ascorbic acid and products of its
decomposition are excreted from the organism via kidneys. In normal conditions
20-30 mg or 113,5-170,3 mkmol of ascorbic acid is excreted per day with urine.
In animal and plant tissues rather
large concentrations of ascorbic acid are present, in comparison with other
water-soluble vitamins; e.g., human blood plasma contains about 1 mg of
ascorbic acid per 100 ml. Ascorbic acid is especially abundant in citrus
fruits, tomatoes, currant, onion, garlic, cabbage, fruits of wild rose, needles
of a pine-tree.
Sources of vitamin C
http://www.youtube.com/watch?v=o3uSmyYSBJ8&feature=related
http://www.youtube.com/watch?v=ACBQQOUcn-Y&feature=related
Vitamin C is obtained through the
diet by the vast majority of the world's population. The richest natural
sources are fruits and vegetables, and of those, the camu camu fruit and the
billygoat plum contain the highest concentration of the vitamin. It is also
present in some cuts of meat, especially liver. Vitamin
C as ascorbic acid is the most widely taken nutritional supplement and is
available in a variety of forms from tablets and drink mixes to pure ascorbic
acid crystals in capsules or as plain powder.
Plant sources
Rose hips are a
particularly rich source of vitamin C Citrus fruits (orange, lemon, grapefruit,
lime), tomatoes, and potatoes are good common sources of vitamin C. Other foods
that are good sources of vitamin C include papaya, broccoli, brussels sprouts,
black currants, strawberries, cauliflower, spinach, cantaloupe, kiwifruit,
cranberries and red peppers. Ascorbic acid in food is largely destroyed by
cooking.
Although the symptoms of scurvy in man can be
prevented by as little as 20 mg of ascorbic acid per day, there are evidences
that far larger amounts may be required for completely normal physiological
function and well-being. Day necessity of vitain C: 50 - 70 mg. But in
different diseases, pregnancy, in hard physical and mental work, in growing
organism, after operations the day requirement of vitamin C increased.
Vitamin P
(bioflavonoids).
This is the group of compounds
(rutin, hesperedin, katecholamines) supporting the elasticity of capillaries,
strengthen their walls and decrease the permeability.
Vitamin P takes part in the
oxidative-reduction processes. It oppresses the activity of enzyme
hyaluronidase protecting the hyaluronic acid which is necessary for elasticity
of vessel walls.
The deficiency of vitamin P in organism results in the
petechias (dot hemorrhages on skin).
Day necessity of vitamin P is not clear exactly (about
25-50 mg). In some diseases 1-