Enzymes
The key element in clinical diagnosis
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. Almost
every significant life process is dependent on enzyme activity.
 Intracellular enzymes catalyze the reactions of metabolic
pathways.
 Plasma membrane enzymes regulate catalysis within cells in
response to extracellular signals.
 Circulatory system enzymes are responsible for regulating
the clotting of blood.
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.).
The enzyme's name is comprised of the names of:
 The substrate(s),
 The product(s) and
 The enzyme's functional class.
In the enzyme acetyl choline esterase for example, 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.
 The substrate of this enzyme is acetyl choline.
 The products are acetate and choline base.
 The enzyme functional clase is esterase because it is
hydrolyze the ester bond in the acetyl choline.
In everyday usage, most enzymes are still called by their common
name.
Number
1.
Classification
Oxidoreductases
Biochemical Properties
Act on many chemical groupings to add
or remove hydrogen atoms.
Transfer functional groups between
donor and acceptor molecules. Kinases
2.
Transferases
are specialized transferases that regulate
metabolism by transferring phosphate
from ATP to other molecules.
3.
Hydrolases
Add water across a bond, hydrolyzing it.
Add H2O, NH3 or CO2 across double
4.
Lyases
bonds, or remove these elements to
produce double bonds.
Carry out many kinds of isomerization: L
5.
Isomerases
to D isomerizations, mutase reactions
(shifts of chemical groups) and others.
Catalyze reactions in which two chemical
6.
Ligases
groups are joined (or ligated) with the
use of energy from ATP.
Enzymes are also classified on the basis of their
composition:
1. Simple enzymes:
They are composed wholly of protein.
2. Complex enzymes:
They are composed of protein plus non-protein component
(a
relatively small organic molecule).
Complex enzymes are also known as HOLOENZYMES.
In this terminology the protein component is known as the
APOENZYME.
Non-protein
component
is
known
as
the
COENZYME
or
PROSTHETIC GROUP.
When the binding between the apoenzyme and non-protein
components is non-covalent, the small organic molecule is called
coenzyme. When the binding between the apoenzyme and nonprotein components is covalent, the small organic molecule is
called Prosthetic group.
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. While those which
have a lower affinity for metal ion, but still require the metal ion
for activity, are known as METAL-ACTIVATED ENZYMES.
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,
Succinic dehydrogenase (SDH) always catalyzes an oxidation
reduction reaction and its substrate is succinic acid.
Alcohol dehydrogenase (ADH) also 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 alcohol
dehydrogenase, 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 Lisomer.
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 Disomers 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.
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. These subunits
combine in various combinations leading to 5 distinct isozymes.
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.
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
million fold, 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.
A
+
B
C+ D
At equilibrium, there is no further apparent change and the rate of
the forward reaction becomes equal to that of the backward one,
hence,
v1 = v2
and
k1 [A] [B] = k2 [C] [D]
[C] [D]
---------- = k1 / k2 = k
[A] [B]
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.
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 combined,
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 <--> EScomplex<--> ES*complex<--> EPcomplex<--> 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:
Km =[S])2-1) = [S]
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 (S) is numerically equal to the Michaelis-Menten
constant (Km) . In this derivation, the units of Km are those used to
specify the concentration of S, usually Molarity.
Plotting of substrate concentration versus reaction velocity in
Michaelis-Menten equation:
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.
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 EScomplex 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:
Take the inverse:
1/v1 = Km /Vmax[S]
+
1/Vmax
Plots of 1/v1 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
Lineweaver-Burk
transformation
of
the
Michaelis-Menton
equation is 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 semiselectively inhibits DNA synthesis of malignant cells.
 The use of aspirin to inhibits the synthesis of prostaglandins
which are at least partly responsible for the aches and pains
of arthritis.
 The use of sulfa drugs to inhibit the folic acid synthesis that
is essential for the metabolism and growth of diseasecausing bacteria.
Enzyme inhibitors fall into two broad classes:
1. Inhibitors causing irreversible inactivation of enzymes.
2. Inhibitors whose inhibitory effects can be reversed.
Irreversible Inhibitors:
They 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 EScomplex . 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:
They can be divided into three categories:
1. Competitive inhibitors.
2. Noncompetitive inhibitors.
3. Uncompetitive inhibitors.
Inhibitor
Type
Binding Site on Enzyme
Kinetic effect
Competitive
Inhibitor
 Specifically at the
catalytic site.
 It competes with
substrate for binding.
 Inhibition is reversible by
substrate.
 Vmax is unchanged.
 Km 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 conc.
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 is decreased.
When the reversible inhibitor concentration drops, enzyme activity
is regenerated. Usually these inhibitors bind to enzymes by noncovalent
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] / [EI 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.
Lineweaver-Burk Plots of Inhibited Enzymes
Enzymes in the Diagnosis of Pathology
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).
 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.
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.
The measurement of LDH is especially diagnostic for myocardial
infarction because this enzyme exist in 5 closely related, but
slightly different forms (isozymes).
METABOLISM
METABOLISM is the set of chemical reactions that occur in living
organisms in order to maintain life. These processes allow
organisms to grow and reproduce, maintain their structures, and
respond to their environments.
Metabolism is usually divided into two categories. CATABOLISM
breaks down large molecules, for example to harvest energy in
cellular respiration. ANABOLISM , on the other hand, uses
energy to construct components of cells such as proteins and
nucleic acids.
The
chemical
reactions
of
metabolism
are
organized
into
metabolic pathways, in which one chemical is transformed into
another by a sequence of enzymes.
Most of the structures that make up animals, plants and microbes
are made from three basic classes of molecule:
1. amino acids
2. carbohydrates
3. lipids
As these molecules are vital for life, metabolism focuses on
making these molecules, in the construction of cells and tissues,
or breaking them down and using them as a source of energy, in
the digestion and use of food.
Many important biochemicals can be joined together to make
polymers such as DNA and proteins. These macromolecules are
essential parts of all living organisms.
CATABOLISM
Catabolism is the set of metabolic processes that break down
large molecules. These include breaking down and oxidizing food
molecules. The purpose of the catabolic reactions is to provide the
energy and components needed by anabolic reactions. The exact
nature of these catabolic reactions differ from organism to
organism.
The most common set of catabolic reactions in animals can be
separated into three main stages:
1. Large organic molecules (proteins, carbohydrates or lipids)
are digested into their smaller components outside cells.
2. These smaller molecules are taken up by cells and converted
to smaller molecules, usually acetyl coenzyme A, which
releases some energy.
3. The acetyl group of CoA is oxidized to H2O and CO2 in the
citric acid cycle and electron transport chain, releasing the
energy that is stored by reducing the coenzyme NAD+
(nicotinamide adenine dinucleotide) into NADH.
Digestion
Macromolecules such as starch, cellulose or proteins cannot be
rapidly taken up by cells and need to be broken into their smaller
units before they can be used in cell metabolism.
Several common classes of enzymes digest these polymers. These
digestive enzymes include proteases that digest proteins into
amino acids, as well as glycoside hydrolazes that digest
polysaccharides into monosaccharides. Lipases digest lipids into
fatty acids and glycerol.
Animals secrete these enzymes from specialized cells in
their
guts. The amino acids or sugars released by these extracellular
enzymes are then pumped into cells by specific active transport
proteins.
Energy from organic compounds
Carbohydrate catabolism is the breakdown of carbohydrates into
smaller units. Carbohydrates are usually taken into cells once they
have been digested into monosaccharides.
Once inside, the major route of breakdown is glycolysis, where
sugars such as glucose and fructose are converted into pyruvate
and some ATP is generated.
Pyruvate is an intermediate in several metabolic pathways, but
the majority is converted to acetyl-CoA and fed into the citric acid
cycle.
Although some more ATP is generated in the citric acid
cycle, the most important product is NADH, which is made from
NAD+ as the acetyl-CoA is oxidized.
An alternative route for glucose breakdown is the pentose
phosphate pathway, which reduces the coenzyme NADPH and
produces pentose sugars such as ribose, the sugar component of
nucleic acids.
Fats are catabolized by hydrolysis to free fatty acids and glycerol.
The glycerol enters glycolysis and the fatty acids are broken down
by beta oxidation to release acetyl-CoA, which then is fed into the
citric acid cycle.
Fatty
acids
release
more
energy
upon
oxidation
than
carbohydrates because carbohydrates contain more oxygen in
their structures.
Amino acids are either used to synthesize proteins and other
biomolecules, or oxidized to urea and carbon dioxide as a source
of energy. The oxidation pathway starts with the removal of the
amino group by a transaminase. The amino group is fed into the
urea cycle, leaving a deaminated carbon skeleton in the form of a
keto acid. Several of these keto acids are intermediates in the
citric acid cycle, for example the deamination of glutamate forms
α-ketoglutarate.
The glucogenic amino acids can also be
converted into glucose, through gluconeogenesis.
ANABOLISM
Anabolism is the set of constructive metabolic processes where
the energy released by catabolism is used to synthesize complex
molecules. In general, the complex molecules that make up
cellular structures are constructed step-by-step from small and
simple precursors.
Anabolism involves three basic stages:
1. The
production
of
precursors
such
as
amino
acids,
monosaccharides, isoprenoids and nucleotides.
2. Their activation into reactive forms using energy from ATP.
3. The assembly of these precursors into complex molecules
such as proteins, polysaccharides, lipids and nucleic acids.
Carbohydrates Metabolism
In carbohydrate anabolism, simple organic acids can be converted
into monosaccharides such as glucose and then used to assemble
polysaccharides such as starch. The generation of glucose from
compounds like pyruvate, lactate, glycerol, glycerate 3-phosphate
and amino acids is called gluconeogenesis.
Gluconeogenesis
converts
pyruvate
to
glucose-6-phosphate
through a series of intermediates, many of which are shared with
glycolysis. However, this pathway is not simply glycolysis run in
reverse, as several steps are catalyzed by non-glycolytic enzymes.
Polysaccharides and glycans are made by the sequential addition
of monosaccharides by glycosyltransferase from a reactive sugarphosphate donor such as uridine diphosphate glucose (UDPglucose)
to
an
acceptor
hydroxyl
group
on
the
growing
polysaccharide.
Glycolysis
Digestion of Dietary Carbohydrates
Dietary carbohydrate from which humans gain energy enter the
body in complex forms, such as disaccharides and the polymers
starch (amylose and amylopectin) and glycogen. The polymer
cellulose is also consumed but not digested. The first step in the
metabolism of digestible carbohydrate is the conversion of the
higher polymers to simpler, soluble forms that can be transported
across the intestinal wall and delivered to the tissues.
Oxidation of glucose is known as glycolysis. Glucose is oxidized to
either lactate or pyruvate. Under aerobic conditions, the dominant
product in most tissues is pyruvate and the pathway is known as
aerobic glycolysis. When oxygen is depleted, as for instance
during prolonged vigorous exercise, the dominant glycolytic
product in many tissues is lactate and the process is known as
anaerobic glycolysis.
The Energy Derived from Glucose Oxidation
Glucose
+
2 ADP + 2 NAD+ + 2 Pi
2 Pyruvate + 2 ATP + 2 NADH + 2 H+
The
NADH
mitochondrial
generated
ATP
during
synthesis
glycolysis
via
is
oxidative
used
to
fuel
phosphorylation,
producing either two or three equivalents of ATP.
The net yield from the oxidation of 1 mole of glucose to 2 moles
of pyruvate is, therefore, either 6 or 8 moles of ATP.
Complete oxidation of the 2 moles of pyruvate, through the Citric
Acid Cycle (TCA cycle) yields an additional 30 moles of ATP; the
total yield, therefore being either 36 or 38 moles of ATP from the
complete oxidation of 1 mole of glucose to CO2 and H2O.
The citric acid cycle is part of a metabolic pathway involved in the
chemical conversion of carbohydrates, fats and proteins into
carbon dioxide and water to generate a form of usable energy.
The Individual Reactions of Glycolysis
The pathway of glycolysis can be seen as consisting of 2 separate
phases.
 The first is the chemical priming phase requiring energy in
the form of ATP.
 The second is considered the energy-yielding phase.
In the first phase,
2 equivalents of ATP are used to convert glucose to fructose 1,6bisphosphate (F1,6BP).
In the second phase,
fructose 1,6-bisphosphate (F1,6BP) is degraded to pyruvate, with
the production of 4 equivalents of ATP and 2 equivalents of NADH.
Pathway of Glycolysis from glucose to pyruvate (Lactate).
Embden-Mayerhof- ‫دورة امدن مايرهوف‬
Enzymes involved in Glycolysis:
1. Hexokinase & Glucokinase:
The ATP-dependent phosphorylation of glucose to form glucose 6phosphate (G6P) is the first reaction of glycolysis, and is catalyzed
by tissue-specific isoenzymes known as hexokinases.
The phosphorylation accomplishes two goals:
 First, the hexokinase reaction converts nonionic glucose into
an anion that is trapped in the cell, since cells lack transport
systems for phosphorylated sugars.
 Second, the otherwise biologically inert glucose becomes
activated into a labile form capable of being further
metabolized.
Four mammalian isozymes of hexokinase are known (Types I IV), with the Type IV isozyme often referred to as glucokinase.
Glucokinase is the form of the enzyme found in hepatocytes.
Non-hepatic tissues, which contain hexokinase rapidly and
efficiently trap blood glucose within their cells by converting it to
glucose-6-phosphate. One major function of the liver is to deliver
glucose to the blood and this is ensured by having a glucose
phosphorylating enzyme glucokinase.
This feature of hepatic glucokinase allows the liver to buffer blood
glucose. After meals, when postprandial blood glucose levels are
high, liver glucokinase is significantly active, which causes the
liver preferentially to trap and to store circulating glucose. When
blood glucose falls to very low levels, tissues such as liver and
kidney, which contain glucokinases but are not highly dependent
on glucose, do not continue to use the meager glucose supplies
that remain available. At the same time, tissues such as the brain,
which are critically dependent on glucose, continue to scavenge
blood glucose.
Under various conditions of glucose deficiency, such as long
periods between meals, the liver is stimulated to supply the blood
with glucose through the pathway of gluconeogenesis. The levels
of glucose produced during gluconeogenesis are insufficient to
activate glucokinase, allowing the glucose to pass out of
hepatocytes and into the blood.
2. Aldolase:
Aldolase catalyses the hydrolysis of F1,6BP into two 3-carbon
products:
dihydroxyacetone
glyceraldehyde
3-phosphate
phosphate
(G3P).
The
(DHAP)
aldolase
and
reaction
proceeds readily in the reverse direction, being utilized for both
glycolysis and gluconeogenesis.
3. Triose Phosphate Isomerase:
The two products of the aldolase reaction equilibrate readily in a
reaction catalyzed by triose phosphate isomerase. Succeeding
reactions of glycolysis utilize G3P as a substrate; thus, the
aldolase reaction is pulled in the glycolytic direction by mass
action principals.
4. Glyceraldehyde-3-Phosphate Dehydrogenase:
The second phase of glucose catabolism features the energyyielding glycolytic reactions that produce ATP and NADH. In the
first
of
these
reactions,
glyceraldehyde-3-P
dehydrogenase
(G3PDH) catalyzes the NAD+-dependent oxidation of G3P to 1,3bisphosphoglycerate (1,3BPG) and NADH. The G3PDH reaction is
reversible, and the same enzyme catalyzes the reverse reaction
during gluconeogenesis.
5. Phosphoglycerate Kinase:
The high-energy phosphate of 1,3-BPG is used to form ATP and 3phosphoglycerate (3PG) by the enzyme phosphoglycerate kinase.
Note that this is the only reaction of glycolysis or gluconeogenesis
that involves ATP and yet is reversible under normal cell
conditions. Associated with the phosphoglycerate kinase pathway
is an important reaction of erythrocytes, the formation of 2,3bisphosphoglycerate, 2,3BPG (see Figure below) by the enzyme
bisphosphoglycerate mutase. 2,3BPG is an important regulator of
hemoglobin's
affinity
for
oxygen.
Note
that
2,3-
bisphosphoglycerate
phosphatase
degrades
2,3BPG
to
3-
phosphoglycerate, a normal intermediate of glycolysis. The
2,3BPG shunt thus operates with the expenditure of 1 equivalent
of ATP per triose passed through the shunt. The process is not
reversible under physiological conditions.
6. Phosphoglycerate Mutase and Enolase:
The remaining reactions of glycolysis are aimed at converting the
relatively low energy phosphoacyl-ester of 3PG to a high-energy
form and harvesting the phosphate as ATP. The 3PG is first
converted to 2PG by phosphoglycerate mutase and the 2PG
conversion to phosphoenoylpyruvate (PEP) is catalyzed by enolase
7. Pyruvate Kinase:
The final reaction of aerobic glycolysis is catalyzed by the highly
regulated enzyme pyruvate kinase (PK). In this strongly exergonic
reaction, the high-energy phosphate of PEP is conserved as ATP.
The loss of phosphate by PEP leads to the production of pyruvate
in an unstable enol form, which spontaneously tautomerizes to
the more stable, keto form of pyruvate. This reaction contributes a
large proportion of the free energy of hydrolysis of PEP.
Anaerobic Glycolysis
Under aerobic conditions, pyruvate in most cells is further
metabolized via the TCA cycle. Under anaerobic conditions and in
erythrocytes under aerobic conditions, pyruvate is converted to
lactate by the enzyme lactate dehydrogenase (LDH), and the
lactate is transported out of the cell into the circulation.
The conversion of pyruvate to lactate, under anaerobic conditions,
provides the cell with a mechanism for the oxidation of NADH
(produced during the G3PDH reaction) to NAD+; which occurs
during the LDH catalyzed reaction.
Aerobic glycolysis generates substantially more ATP per mole of
glucose oxidized than does anaerobic glycolysis.
The utility of anaerobic glycolysis, to a muscle cell when it needs
large amounts of energy, stems from the fact that the rate of ATP
production from glycolysis is approximately 100X faster than from
oxidative phosphorylation.
Pyruvate Metabolism
Pyruvate is the branch point molecule of glycolysis. The ultimate
fate of pyruvate depends on the oxidation state of the cell. In the
reaction catalyzed by G3PDH a molecule of NAD+ is reduced to
NADH.
In order to maintain the re-dox state of the cell, this NADH must
be re-oxidized to NAD+.
During aerobic glycolysis this occurs in the mitochondrial electron
transport chain generating ATP. Thus, during aerobic glycolysis
ATP is generated from oxidation of glucose directly at the PGK and
PK reactions as well as indirectly by re-oxidation of NADH in the
oxidative phosphorylation pathway. Additional NADH molecules
are generated during the complete aerobic oxidation of pyruvate
in the TCA cycle.
Pyruvate enters the TCA cycle in the form of acetyl-CoA which is
the product of the pyruvate dehydrogenase reaction. The fate of
pyruvate during anaerobic glycolysis is reduction to lactate.
LACTATE METABOLISM
During anaerobic glycolysis, the large quantity of NADH produced
is oxidized by reducing pyruvate to lactate. This reaction is carried
out by lactate dehydrogenase, (LDH).
The lactate produced during anaerobic glycolysis diffuses from the
tissues and is transproted to highly aerobic tissues such as cardiac
muscle and liver. The lactate is then oxidized to pyruvate in these
cells by LDH and the pyruvate is further oxidized in the TCA cycle.
If the energy level in these cells is high the carbons of pyruvate
will be diverted back to glucose via the gluconeogenesis pathway.
FRUCTOSE METABOLISM
Diets containing large amounts of sucrose can utilize the fructose
as a major source of energy. The pathway to utilization of fructose
differs in muscle and liver.
In the muscle, which contains only hexokinase can phosphorylate
fructose to F6P which is a direct glycolytic intermediate.
In the liver, which contains mostly glucokinase
(specific for
glucose as its substrate) requires the function of additional
enzymes (aldolases) to utilize fructose in glycolysis.
Clinical Significances of Fructose Metabolism
FRUCTOSURIA is metabolic disorder caused by the lack of
fructokinase.
HEREDITARY FRUCTOSE INTOLERANCE
is a potentially lethal
disorder resulting from a lack of aldolase B. The disorder is
characterized by severe hypoglycemia and vomiting following
fructose intake. Prolonged intake of fructose by infants with this
defect leads to vomiting, poor feeding, jaundice ‫يرقاان وو عارور‬
‫باالعاازازا‬, hepatomegaly, hemorrhage and eventually hepatic
failure and death.
Patients remain symptom free on a diet devoid of fructose and
sucrose.
GALACTOSE METABOLISM
Galactose, which is metabolized from the milk sugar, lactose
enters glycolysis by its conversion to glucose-1-phosphate (G1P).
This occurs through a series of steps summarized in the following
figure:
Clinical Significances of Galactose Metabolism
GALACTOSEMIA is a major symptom of the loss of the two
enzymes,
galactokinase
and
galactose-1-phosphate
uridyl
transferase.
Vomiting and diarrhea occur following ingestion of milk, hence
individuals are termed lactose intolerant. Clinical findings elevated
blood galactose, hyper galactosemia, urinary galactitol excretion
and hyper amino acid uria.
Unless controlled by exclusion of galactose from the diet, these
galactosemias can go on to produce blindness and fatal liver
damage.
GLYCOGEN METABOLISM
The body obtains glucose from either one of the following:
1. Directly from the diet
2. From amino acids and lactate via gluconeogenesis.
Glucose obtained from these two primary sources either remains
soluble in the body fluids or is stored in a polymeric form,
glycogen.
Glycogen is considered the principal storage form of glucose and is
found mainly in liver and muscle. With up to 10% of its weight as
glycogen, the liver has the highest specific content of any body
tissue.
Muscle has a much lower amount of glycogen per unit mass of
tissue, but since the total mass of muscle is so much greater than
that of liver, total glycogen stored in muscle is about twice that of
liver.
Stores of glycogen in the liver are considered the main buffer of
blood glucose levels.
GLYCOGENOLYSIS
Degradation of stored glycogen, termed glycogenolysis, occurs
through the action of glycogen phosphorylase.
The glucose-1-phosphate produced by the action of phosphorylase
is converted to glucose-6-phosphate by phosphor gluco mutase.
The enzyme phosphate is transferred to C-6 of glucose-1phosphate generating glucose-1,6-phosphate as an intermediate.
The conversion of glucose-6-phosphate to glucose, which occurs
in the liver, kidney and intestine, by the action of glucose-6phosphatase does not occur in skeletal muscle as these cells lack
this enzyme. Therefore, any glucose released from glycogen stores
of muscle will be oxidized in the glycolytic pathway. In the liver
the action of glucose-6-phosphatase allows glycogenolysis to
generate free glucose for maintaining blood glucose levels.
Gluconeogenesis
Gluconeogenesis is the biosynthesis of new glucose, (i.e. not
glucose from glycogen).
The production of glucose from other metabolites is necessary for
use as a fuel source by the brain, testes, erythrocytes and kidney
medulla since glucose is the sole energy source for these organs.
During starvation, however, the brain can derive energy from
ketone bodies which are converted to acetyl-CoA.
Substrates for Gluconeogenesis
Lactate:
Lactate is a predominate source of carbon atoms for glucose
synthesis by gluconeogenesis. During anaerobic glycolysis in
skeletal muscle, pyruvate is reduced to lactate by lactate
dehydrogenase (LDH).
LDH
Pyruvate
Lactate
This reaction serves two critical functions during anaerobic
glycolysis:
1. LDH reaction requires NADH and yields NAD+ which is then
available
for
use
by
the
glyceraldehyde-3-phosphate
dehydrogenase reaction of glycolysis.
2. The lactate produced by the LDH reaction is released to the
blood stream and transported to the liver where it is
converted to glucose. The glucose is then returned to the
blood for use by muscle as an energy source and to replenish
glycogen stores. This cycle is termed the Cori cycle.
Pyruvate:
Pyruvate, generated in muscle and other peripheral tissues, can be
transaminated to alanine which is returned to the liver for
gluconeogenesis.
The transamination reaction requires an α-amino acid as donor of
the amino group, generating an α-keto acid in the process. This
pathway is termed the
glucose-alanine cycle.
The glucose-alanine cycle is, therefore, an indirect mechanism for
muscle to eliminate nitrogen while replenishing ‫ يازود‬its energy
supply. However, the major function of the glucose-alanine cycle
is to allow non-hepatic tissues to deliver the amino portion of
catabolized amino acids to the liver for excretion as urea. Within
the liver the alanine is converted back to pyruvate and used as a
gluconeogenic substrate (if that is the hepatic requirement) or
oxidized in the TCA cycle. The amino nitrogen is converted to urea
in the urea cycle and excreted by the kidneys.
Citric Acid Cycle
The citric acid cycle — also known as the tricarboxylic acid cycle
(TCA cycle), the Krebs cycle, is a series of enzyme-catalysed
chemical reactions of central importance in all living cells that use
oxygen as part of cellular respiration. In eukaryotes, the citric acid
cycle occurs in the matrix of the mitochondrion. In aerobic
organisms, the citric acid cycle is part of a metabolic pathway
involved in the chemical conversion of carbohydrates, fats and
proteins into carbon dioxide and water to generate a form of
usable energy. Other relevant reactions in the pathway include
those in glycolysis and pyruvate oxidation before the citric acid
cycle, and oxidative phosphorylation after it. In addition, it
provides precursors for many compounds including some amino
acids and is therefore functional even in cells performing
fermentation.
The TCA cycle showing enzymes, substrates and products. The
GTP generated during the succinate thiokinase (succinyl-CoA
synthetase) reaction is equivalent to a mole of ATP by virtue of the
presence of nucleoside diphosphokinase. The 3 moles of NADH
and 1 mole of FADH2 generated during each round of the cycle
feed into the oxidative phosphorylation pathway. Each mole of
NADH leads to 3 moles of ATP and each mole of FADH2 leads to 2
moles of ATP. Therefore, for each mole of pyruvate which enters
the TCA cycle, 12 moles of ATP can be generated. IDH = isocitrate
dehydrogenase. α-KGDH = α-ketoglutarate dehydrogenase. MDH
= malate dehydrogenase. Place mouse over cycle intermediate
names to see their structures.
CITRIC ACID CYCLE (TCA CYCLE)
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