UNIT 9

MULTIENZYME
COMPLEXES
Structure
9.1 Introduction 9.6 Summary
Objectives 9.7 Terminal Questions
9.2 Multienzyme Complexes as 9.8 Answers
Regulatory Enzymes
9.9 Suggested Readings
9.3 Occurrence and Isolation
9.4 Phylogenetic Distribution
and Properties
Pyruvate Dehydrogenase Complex
Fatty Acyl Synthase Complex

9.5 Isoenzymes
Lactate Dehydrogenase (LDH) Isoenzyme
Physiological Role of Isoenzymes
Pharmacological Role of Isoenzymes

9.1 INTRODUCTION
If you are given two choices- a single enzyme or an enzyme complex with two
or more enzymes, what do you think will be suitable to study about the
structure, kinetics and mechanism of action of enzymes? Single enzyme can
be studied more easily because it will be free from any interference from
competing reactions. But now you should look into the situation in an intact
cell. Enzymes in a cell do not act as single isolated enzyme doing catalysis of
reaction with single or multiple substrates in buffer solutions. It means that the
operation of enzyme in vivo depends on the nature of conditions of cell and are
more likely to be different then its functioning in vitro. In an intact cell with
different subcellular membranous structures, several enzymes compete with
one another for substrates and effectors. Therefore it is very much likely that
the enzymes are physically associated with other enzymes to different extent.
You will also notice that organized system of cell enzyme system is more
complex. If you will recall from unit (Unit-1, Block-1) about the Enzyme
Commission for nomenclature of enzymes, enzymes are classified on the
basis of catalysis of reaction and not on their physical organization. Let us
introduce a new term, multienzyme complex- stable gathering of several
enzymes with multiple catalytic domains. In this unit we will read more about
these complexes to understand the regulation of enzyme activity. 125
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Objectives
After studying this unit, you will be able to:
 define multi enzyme complexes;
 determine their role as regulatory enzymes;
 discuss the major role played by key multienzyme complexes;
 enlist their properties; and
 explain the function of isoenzymes.

9.2 MULTIENZYME COMPLEXES AS

REGULATORY ENZYMES
In free solution, the rate of enzyme catalyzed reaction is dependent on the
enzyme concentration and the substrate concentration. In a metabolic
pathway, the product of one reaction acts as the substrate for the next enzyme
to act upon in the pathway. An enzyme- catalyzed reaction is said to be
diffusion- limited at suboptimal concentrations, since it is dependent on the
random collisions of enzyme and substrate. The direct metabolite transfer
from one enzyme to other avoids dilution of the metabolite in aqueous
environment of the cell and the rate of reaction would increase.

In the cells, for such metabolic channeling to occur, the enzymes of a certain
pathway are spatially organized. Some enzymes associate with other
enzymes involved in a certain pathway to give multienzyme complexes. For
enzymes participating in such complexes, the substrate diffusion is not rate-
limiting. In eukaryotes, most enzymes don’t diffuse freely in cytoplasm but are
concentrated in particular parts of the cell beside other enzymes or proteins of
related process.

A multienzyme complex is a stable assembly of more than one enzyme, which

are generally involved in sequential catalytic transformations. These are
different from multienzyme polypeptides, which have multiple catalytic domains
found in single polypeptide. Such examples include: pyruvate dehydrogenase,
a complex of three different enzymes that collectively catalyze the oxidation of
pyruvate into acetyl-CoA by the process of pyruvate decarboxylation; fatty acyl
CoA synthase, a complex of six enzymes that assemble in the shape of a
barrel to generate fatty acids.

9.3 OCCURRENCE AND ISOLATION

During metabolic processes, a number of enzymes catalyze the sequence of
reactions in such a way that the product of one enzyme- catalyzed reaction
becomes the substrate for the next enzyme as shown in the following
sequence.

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The before-mentioned reaction represents a sequential metabolic pathway in
which S1, S2, S3 and S4 are the substrates for the enzymes E1, E2, E3 and
E4. The overall rate of reaction for conversion of S1 to S5 will depend fairly on
the coordination between these four enzymes. It has been known that for
some metabolic pathways, certain enzymes are physically associated with
each other to form multienzyme complex.

The isolation and characterization of multienzyme complexes is

comparatively more difficult than that of a single enzyme for example-
pyruvate dehydrogenase complex and yeast fatty-acid synthase complex.
Isolation of multienzyme complex, yeast fatty- acid synthase was found to be
difficult as it involves separation of the polypeptide chains for the seven
catalytic activities of the whole enzyme. Later it was revealed that the whole
complex consists of two multifunctional polypeptide chains and the smaller
fragments observed earlier were due to limited proteolysis during isolation of
the enzyme complex. For another such complex- pyruvate dehydrogenase
complex, it is well established that it comprises of three different enzyme
activities, it has been difficult to determine the exact number of polypeptide
chains present in the complex. The reason might be (a) disparity in isolation
procedures yielding complexes with slightly different compositions (b) intact
cell complexes with slight difference in composition also exist (c)
dissociation during isolation. The existence of a multienzyme complex can
be deduced when a component of the enzyme complex is being isolated and
also found to be purified along with another enzyme from the same metabolic
pathway. The presence of a multienzyme complex can further be confirmed
if the ratio of the enzyme activities remains constant during isolation e.g.
carbamoyl phosphate synthase, aspartate carbamoyltransferase, dihydro-
orotase (CAD). Examples include enzymes having kinase and phosphatase
activities present in the same polypeptide is isocitrate dehydrogenase kinase
and isocitrate dehydrogenase phosphatase.

SAQ 1
Do as Directed:

a) In the cells, for metabolic channeling to occur, the enzymes

of a certain pathway are spatially organized. (True/False)

b) A multienzyme complex is an unstable/stable assembly of

more than one enzyme. (Pick one option)

c) The isolation of multienzyme complexes is comparatively

simpler than that of a single enzyme. (True/False)

d) A multienzyme complex is similar to multienzyme

polypeptide. (True/False)
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9.4 PHYLOGENETIC DISTRIBUTION AND

PROPERTIES
Generally, multienzyme polypeptides and multienzyme proteins are both
more complex and more common in eukaryotes as compared to
prokaryotes. This can be observed with the examples of fatty acid synthase,
pyruvate dehydrogenase and the enzymes of tryptophan biosynthesis. If you
can recall from your school textbooks the difference between prokaryotes
and eukaryotes, you will understand that even after the intracellular
compartmentalization, the volume of the eukaryotic cell in which free
diffusion of substrate and enzyme can occur is much greater than a
prokaryotic cell. Therefore, if the intracellular concentration of catalytic
centers and substrates are similar in both types of cells, even then the
diffusion time is possibly rate limiting in eukaryotes. Available information on
multienzyme complexes such as carbamoyl phosphate synthase, aspartate
carbamoyl transferase, fatty acid synthase and several others suggest that
evolution process consequently leads to tightly associated multienzyme
complexes. Genetic evidence also points out clustering or fusion of genes
coding for these proteins.

Multienzyme complexes exhibit characteristic properties. Less time will be

required for a product of one enzyme to diffuse to the catalytic site of the next
enzyme in the case of a physically associated multienzyme. There are two
instances in which transit time plays an important role (a) when the
intermediate has short half-life e.g. the carbamoyl phosphate probably has a
short half- life, because there are enzymes capable of degrading it. Its
association with aspartate carbamoyl transferase, probably prevents its
degradation (b) when intermediate has high Mr and hence diffuse slowly e.g.
the second step in the reaction sequence of the pyruvate dehydrogenase
system involves two enzyme bound intermediates hydroxylethyl-
thiaminepyrophosphate-pyruvate dehydrogenase and dihydrolipoamide
acetyltransferase.

In addition to the transit time, the transient time i.e. the time required to change
from one steady- state to another also gets reduced in a multienzyme protein.
Let us consider a system with two enzymes (E1 & E2) :

In a steady state in the cell, if the concentration of A is increased then in

multienzyme protein, the transient time i.e the time required to reach the new
steady- state will be much less.

Multienzyme complex system also makes channeling or compartmentalization

possible. After the formation of an intermediate in the multienzyme protein, it is
not available for the other enzymes outside the complex to be acted upon. This
means that the concentration of such metabolites inside the cell will be lower.
The solvent capacity of water is limited due to the relatively high concentrations
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of solute in the cell compartments. Lowering the concentrations of metabolites
would spare this capacity. The loss of pathway flux by leakage or instability of
free intermediates would also be reduced by channeling.

Coordinate activation of whole multienzyme protein is also observed evidently.

There is possibility that one domain may exert an allosteric effect on adjacent
domain as in the ribulose bisphosphate carboxylase- oxygenase which acts
together along with four other enzymes namely phosphoribulokinase,
phosphoglycerate kinase, phosphoribose isomerase and glyceraldehyde
phosphate dehydrogenase to catalyze five successive reactions of the
Benson- Calvin cycle. The free enzyme is made up of small (S) and large (L)
subunits possessing the structure L8S8, but when available as the five enzyme
complex has a L2S4 structure. Another multienzyme is the arom complex in
which substrate of the first enzyme has the ability to activate all five catalytic
activities of the whole multienzyme protein.

The tryptophan synthase system and pyruvate dehydrogenase system are

examples of multienzyme complexes whereas fatty acid synthase and the
arom complex are the examples of multienzyme polypeptides.

SAQ 2
Fill in the blanks.

a) Multienzyme polypeptides and multienzyme proteins are both more

complex and more common in eukaryotes as compared to .................

b) The volume of the eukaryotic cell in which free diffusion of substrate and
enzyme can occur is ................. than in a prokaryotic cell. [more/less]

c) ................. time is required for a product of one enzyme to diffuse to the

catalytic site of the next enzyme in multienzyme complex. [more/less]

d) ................. reduces loss of pathway flux by leakage or instability of free

intermediates.

9.4.1 Pyruvate Dehydrogenase Complex

Pyruvate dehydrogenase was the first enzyme to be purified. Earlier, in 1950s

it was known that the oxidation of pyruvate was catalyzed by large
homogenous enzyme preparation and that the reaction involves more than one
catalytic step. The system catalyzes an important step regulating the flow of
acetyl groups into the tricarboxylic acid (TCA) cycle. At that time, it was
understood that 2 oxoglutarate dehydrogenase was closely related to pyruvate
dehydrogenase catalyzing an oxidative decarboxylation and has same cofactor
requirements. It also catalyzes a step in TCA cycle. In the mid-1970s a third
multienzyme system was identified which was capable of oxidizing branched
chain keto acids derived from amino acids valine, isoleucine and leucine.
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These three multienzyme complexes, branched chain oxoacid dehydrogenase,
2- oxoglutarate dehydrogenase and pyruvate dehydrogenase display common
properties, as they:

a) employ the same five cofactors, lipoate, coenzyme A, FAD, NAD+ and
thiamine pyrophosphate,
b) demonstrate structural and mechanical similarities, with a transacylase at
the core of the complex and dehydrogenase and decarboxylase on the
edge,
c) have three catalytic centres catalyzing a dehydrogenation, a
decarboxylation and a transacylation,
d) share similar dihydrolipoamide dehydrogenase component excluding in
Pseudomonas putida.
Apart from sharing similarities, these complexes also vary from each other, as:
a) some are based on icosahedral symmetry, others on octahedral,
b) a supplementary polypeptide component is there in mammals, yeast and
probably other eukaryotes, which is vital for appropriate assembly and
functioning of the complex,
c) the decarboxylase constituent may consist of homodimers (2) or
heterotetramers (2 2) of two polypeptides,
d) the catalytic property of some eukaryote complexes is regulated by
phosphorylation of the decarboxylase.
The complex enables pyruvate to move in the TCA cycle, by catalyzing its
decarboxylation. It also utilizes another coenzyme lipoic acid for the oxidation
step and lastly, coenzyme A (CoASH) which reacts to the acetyl lipoamide
complex, producing acetyl CoA as the product.
Pyruvate + CoASH + NAD+  acetyl-CoA + CO2 + NADH
Lester Reed and coworkers, in 1968 reported that the [Link] pyruvate
dehydrogenase multienzyme complex consists of 60 polypeptide chains
having a molecular weight of about 4,600,000. The complex comprises of:
pyruvate dehydrogenase (E1); dihydrolipoyl acetyltransferase (E2) and
dihydrolipoyl dehydrogenase (E3). The catalytic reaction takes place with
enzyme bound substrate, which may be directly or via cofactors thiamine
pyrophosphate (TPP) and lipoate. TPP is associated with E1 and the side
chain of lipoate is covalently bound to lysyl residue of E2. FAD acts as the
prosthetic group for E3. The enzyme complex is about 300 Angstroms in
diameter and may easily undergo dissociation because of being held by non-
covalent interactions. The cubical core complex comprises of 24 subunits of
E2 associated as trimers around which there is a symmetrical arrangement of
E1 and E3. A dimer of E1 and E3 is present on each of the 12 edges and 6 faces
of the cube respectively.
Apart from [Link], the complex has also been studied in various other
organisms and tissues which include: Bacillus stearothermophilus,
130 Azotobacter vinelandii, Pseudomonas spp, Saccharomyces cerevisviae,
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Arabidopsis, Neurospora crassa, Enterococcus faecalis, mammalian heart,
kidney liver and avian tissues. It has been cloned and sequenced because it
plays an important role in genetic deficiencies.
If you can recall from the school text books, you will remember that the
pyruvate dehydrogenase multienzyme complex links glycolytic pathway with
the tricarboxylic acid cycle. How the activity of this multienzyme complex in
prokaryotes [Link] can be controlled or regulated? The answer is the product
acetyl CoA formed in the reaction catalyzed by pyruvate dehydrogenase
complex. Acetyl- CoA is competitive inhibitor with respect of CoA and another
coenzyme NADH (Fig. 9.1). Both NADH and acetyl-CoA may also hinder
acetylation of the bound lipoamide. The pyruvate dehydrogenase complex is
also inhibited by GTP and activated by nucleoside monophosphates. The
regulation in the mammalian system is similar but much complex (Fig. 9.2):
covalent modification is involved while kinase and phosphatase enzymes are
present within the complex. The kinase bound to E2, catalyze a serine residue
phosphorylation in the E1 which inactivates the complex when the intracellular
ratio of [ATP]/[ADP] is high. The kinase is inhibited by ADP and pyruvate and
activated itself by acetyl-CoA and NADH. The phosphatase is activated by Ca 2+
and Mg2+which is responsible for the removal of this phosphate.

Fig. 9.1: Regulation of pyruvate dehydrogenase complex in [Link]

Fig. 9.2: Regulation of mammalian pyruvate dehydrogenase complex

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Isolation and separation of subunits of pyruvate dehydrogenase
multienzyme complex

Fig. 9.3: Isolation of pyruvate dehydrogenase multienzyme complex from [Link]

cells.

The enzyme is often expressed as ‘housekeeping enzyme’ that catalyzes a

fundamental metabolic pathway being managed in almost all cell types and
therefore present in reasonable quantity inside the cells and tissues. The
process for the isolation and purification of this enzyme complex are based on
the formerly developed procedures for the [Link] complex (See Fig: 9.3) or the
132
process of poly (ethylene glycol) fractionation followed by isoelectric
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precipitation and gel filtration adopted for mammalian pyruvate dehydrogenase
complex. The genes for human and Saccharomyces pyruvate dehydrogenase
have been cloned and expressed in [Link] enabling large quantities of the
complex to be isolated and purified for structural studies. The isolated enzyme
can be dissociated by use of 4 mol dm-3 urea, calcium phosphate gel and high
pH or alternatively by using high salt concentrations. The complex can again be
reconstituted from the subunits and the whole enzyme activity can be restored.
The dissociation and reconstitution studies reveal that E 2 has binding sites for
both E1 and E3 but E1and E3 do not bind when E2 is absent. The Mr value of the
polypeptide chains can be predicted by conventional methods and in many
cases, have been established from those inferred by the DNA sequence. In
yeast and mammalian pyruvate dehydrogenase complex, an additional
polypeptide X has been found attached to the E2 core after E1 and E3 have
been dissociated. Chaotropic agents such as urea is required to dissociate the
E2-X subcomplex. The polypeptide X is structurally similar to the N-terminal
region of E2, but differs in C-terminal region.

9.4.2 Fatty Acyl Synthase Complex

Organisms have the ability to synthesize long- chain fatty acids and show
similar basic metabolic pathway, though certain modifications do exist. The
synthesis of fatty acids occurs from acetyl CoA and malonyl CoA as the
precursor and the overall process of acyl chain extension occurs by addition of
two methylene groups and requires transacylations, two reductions, a
dehydration and a condensation and thus involves six catalytic activities. For
the formation of malonyl CoA, an additional enzyme acetyl CoA carboxylase is
necessary for the catalysis of the reaction.

The predominant chain length of fatty acids synthesized and the mechanism of
release of the long- chain acyl carboxylate varies among organisms, but the
sequence of enzyme reaction is alike in all cases. Despite the similarities, the
organization of the enzyme system may be different.

There are two principal classes of fatty acid synthases:

In many animal tissues, avian tissues, Neurospora, and in some prokaryotes

fatty-acid synthase system is found to exist as an aggregated multienzyme
protein. This type of system is referred to as type I fatty acid synthase system.
It was thought that these multienzyme proteins comprise of several tightly
bound polypeptides with each chain having a catalytic activity, but with proper
purification, in which proteolysis was avoided, revealed that they exist as
multienzyme polypeptides. The type 1 fatty- acid synthase may further be
divided on the basis of their size as type IA, having Mr of about 500 000 and
subunit structure 2, and type IB, having Mrvalue from 1 x 106 to 2.4 x 106 and
subunit structure 6-8 or 6 6. The type IA is found in animal tissues and
type IB occur in fungi, algae and certain bacteria such as Corynebacteria,
Mycobacteria and Streptomyces. Fig. 9.4 displays a model proposed for the
organization of domains in type I fatty acid synthase. 133
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Fig. 9.4: Proposed domain structure of animal fatty acid synthase (type 1A)

type II is found in archaea and bacteria, and is characterized by the use of

discrete, monofunctional enzymes for fatty acid synthesis. In [Link] six
separate enzymes: ACP acetyltransferase, ACP malonyltransferase, 3-oxoacyl
ACP synthase, 3-oxoacyl ACP reductase, crotonyl ACP hydratase and enoyl
ACP reductase, together with the acyl carrier protein have been isolated and
purified. Altogether, these enzymes are known to catalyze fatty- acid synthesis
and there is no evidence of their physical association with one another. This
non-aggregated type of system is also known as type II fatty acid synthase
system. A similar system has been found in other bacteria, Phormidium
luridum, Euglena, Avocardo mesocarp, Chlamydomonas and in the chloroplast
from lettuce and spinach.

The mechanism of FAS I and FAS II elongation and reduction is the same, as
the domains of the FAS II enzymes are basically homologous to their domain
counterparts in FAS I multienzyme polypeptides. However, the differences in
the organization of the enzymes - integrated in FAS I, discrete in FAS II - gives
rise to many significant biochemical differences.

The evolutionary history of fatty acid synthases is very much intertwined with
that of polyketide synthases (PKS). Polyketide synthases use a related
mechanism and homologous domains to generate secondary metabolite lipids.
Furthermore, polyketide synthases also display a Type I and Type II
organization. FAS I in animals is thought to have arisen through modification of
PKS I in fungi, whereas FAS I in fungi and the CMN group of bacteria
(corynebacteria, mycobacteria, and nocardia) seem to have arisen separately
through the fusion of FAS II genes.

The yeast fatty- acid synthase system has been systematically explored from
a genetic as well as biochemical standpoint. Genetic mapping has revealed
that there are two unlinked genetic loci on the chromosomes designated as fas
134 1 and fas 2, are accountable for the whole fatty- acid synthase system.
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Modification of its free amino groups using maleic anhydride results in
reversible dissociation of the fatty- acid synthase.

SAQ 3
State whether following statements are True or False:

a) Acetyl- CoA acts as a competitive inhibitor of CoA for regulating

the activity of pyruvate dehydrogenase complex. (True/False).

b) In pyruvate dehydrogenase complex, E2 subunit, catalyze a

serine residue phosphorylation in the E1 which inactivates
the complex when the intracellular ratio of [ATP]/[ADP]
is low. (True/False).

c) In type II fatty acid synthase system, six separate enzymes

known to catalyze fatty - acid synthesis are physically
associated with each other. (True/False).

d) Type I fatty acid synthase system are multienzyme

polypeptides. (True/False).

9.5 ISOENZYMES
A surprising discovery in biochemistry was the finding that a single enzyme
can occur in several different forms within a single tissue. Markert and Møller
proposed that ‘multiple enzyme forms’ in a single species should be known as
isoenzymes or isozymes. However, numerous biochemists felt that the term
‘isoenzymes’ should be limited to those forms rising from genetic control of
primary structure of protein. Due to the differences in opinions, it was finally
recommended that:

The term ‘multiple forms of enzymes’ should be used as a wide term which
covers all proteins occurring naturally in single species and also catalyzing the
same reaction.

The term ‘isoenzymes’ or ‘isozymes’ should be applicable to only those various

forms of enzymes arising from genetically determined disparity in protein
primary structure but not to those which are resulting by alteration of the same
primary sequence. These isozymes may be present in the identical species, in
the similar tissues or even in the same cell. These isozymes usually vary in
the kinetic nature or mode of regulation or usage of cofactor or even in their
subcellular distribution.

Nomenclature of isoenzymes

In 1964, it was also recommended that based on the electrophoretic mobility,

the individual isoenzymes should be numbered and distinguished, with the
number 1 representing the form possessing highest mobility towards anode.
Out of all the other criteria available to specify the properties of isoenzymes
such as chromatography, electrophoresis, chemical structure, kinetics etc. 135
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electrophoresis is the most widely accepted criteria due to the following
reasons:

a) In electrophoresis, the resolution of individual proteins is not improperly

inclined by their application in the original state, as tissue homogenate, or
likewise.

b) The degree of resolution by electrophoresis is generally more effective

than any other approaches of protein separations.

c) It tenders broad applicability and rapidity.

Although, kinetic data are helpful to explore the enzyme multiplicity, they are not
competent to present evidence on the degree of heterogeneity. With respect to
chemical structures, the final goal is to define the interrelationships of
enzymes’ multiple forms in chemical terms, and it is not useful to convey
general approvals of nomenclature of isoenzyme based on structural data
because such data is obtainable for a smaller number of enzyme systems.
Once, structural data becomes available, notations in upper case roman
letters, as assigned for aldolase and lactate dehydrogenase, may be used.

Therefore, it was recommended that:

(a) The normal enzyme name followed by a number should be used, which
should be allotted successively on the basis of their electrophoretic
mobility under definite conditions. The lower number being given to the
forms having high mobility in the direction of anode.

(b) In a complex isoenzyme pattern, the number may be used to represent

major groups, with lower case alphabets in subscript applied serially for
individual subzones (1a 1b 1c 2a 2 b).

(c) Additional characteristics such as molecular weight, subunit structure,

stability should be provided. Subunits may be represented by Roman
upper-case letters or lower-case Greek letters.

The most widely studied isoenzyme is lactate dehydrogenase (LDH), which has
been investigated in numerous laboratories. Several studies have provided a
rich catalogue of data on the biological and clinical significance of LDH.

9.5.1 Lactate Dehydrogenase (LDH) isoenzyme

LDH of human tissues can be separated into five distinct forms by the
application of starch gel electrophoresis. LDH-1 is closest to the anode and
LDH-5 is nearest to the cathode. In adult human liver and muscle, LDH-5 is the
dominant forms whereas, in kidneys and heart, the dominant forms are LDH-1,
LDH-2, LDH-3. Examination of a variety of human tissues has revealed that the
isoenzyme pattern in each case is unique.

After the elucidation of the structure of LDH isoenzyme, it appears that each
isoenzyme consists of four polypeptide chains which are arranged at random
136 from two separate polypeptide subunits A and B. The polypeptide composition
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of each isoenzyme can be represented as:

L DH 1 B B B B ( A 0B 4)

L DH 2 A B B B ( A 1B 3)

L DH 3 AABB ( A 2 B 2 )

L DH 4 AAAB ( A 3 B 1 )

L DH 5 AAAA ( A 4 B 0 )

This formulation was gained by a series of experiments. Firstly, it was shown

that each isoenzyme has a molecular weight of 135,000 and can bind four
molecules of DPN (diphosphopyridine nucleotide). When proteolytic reagents
were added to the solutions, the molecules split into four polypeptide chains of
equal lengths (M.W. 34,000), and these can be separated by electrophoresis
into two distinct forms A and B. Finally, an extensive analysis of tissues from
many sources revealed five isoenzymes almost in all of them. The
arrangement of mixture of two kinds of subunits A and B, into five different
molecules, each containing four subunits was elucidated by Markert (1963).
The molecular structures of LDH and hemoglobin share many similarities.
Each molecule is a tetramer comprising of four polypeptide chains, and each
chain is associated with a catalytic site. Another important difference among
isoezymes is the manner in which the molecules are assembled from their
subunits.

Shaw and Barto (1963) observed the genetic evidence for the subunit
hypothesis for LDH isoenzyme synthesis. They discovered an electrophoretic
variant of polypeptide B in the deer mouse, and were able to show that the
production of A and B subunits is managed by two different non- allelic genes.
Further experiments showed that similar genetic mechanism control the LDH
isoenzyme synthesis in human tissues. Pedigrees listed by Kraus and Neely
(1964) suggested that the LDH variants are inherited as autosomal codominant
characters. If only two genetic loci are involved in the synthesis of the LDH
isoenzyme, then it is hard to interpret the presence of a sixth isoenzyme ‘band
X’, in post-pubertal testis and sperm. The dissociation and recombination
experiments suggest that the ‘band X’ isoenzyme is a tetramer composed of
polypeptides different from A and B. Therefore, the complement of LDH
isoenzyme can be elucidated on the basis of the activity of gene at three loci A,
B and C where each of them is accountable for synthesis of corresponding
polypeptide.

9.5.2 Physiological Role of Isoenzymes

There were apprehensions regarding the existence of five forms of the enzyme
when each of them catalyzes the same reaction. The explanation is based on
the pathway of glycolysis. During the breakdown of glucose, the fate of
pyruvate can be (1) conversion to lactate, and (2) conversion to carbon dioxide
and water. In the skeletal muscles, the energy results from anaerobic
glycolysis with the generation of ATP. Reduced diphosphopyridine nucleotide;
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(DPNH) is also produced during ATP production, and if DPNH does not get
dehydrogenated to DPN, glycolysis would stop. The chief reaction for oxidation
of DPNH is the transformation of pyruvate to form lactate, thus, it is essential
that LDH of skeletal muscle should be able to handle the huge quantity of
pyruvate formed at the time of strong muscular activity. Whereas, in the heart
tissue, there is less requirement for anaerobic energy, maximum amount of the
pyruvate enters citric acid cycle. A particular type of LDH in heart is the one
which is inhibited by small amounts of pyruvate. The major isoenzymes in
heart, comprising mainly polypeptide B, are powerfully inhibited by low
concentrations of pyruvate and permits the metabolism of pyruvate to proceed
via citric acid cycle. On the contrary, the muscle enzyme, having polypeptide A
is inhibited only by very high concentrations of pyruvate, which favors the
transformation of pyruvate to lactate. Therefore, it is ostensible that the
catalytic features of the isoenzymes are apt according to metabolic needs of
the respective tissues.

Wilson, Cahn and Kaplan (1962) studied the LDH isoenzyme present in birds
and breast muscles. Birds which fly infrequently like grouse, pheasants and
domestic fowls have largely LDH-5. Their patterns are like human skeletal
muscle. While performing muscular activities, lactate gets accumulated with
frequent activity fatigue arises. LDH-1 is key isoenzyme in breast muscles of
hummingbird and storm petrel bird which excel in their ability for long and
persistent flights.

In the fetal mouse, there is a dominance of isoenzymes in the LDH-5 end of

the spectrum. This condition allows the tissue function in relatively anaerobic
environment. In chick embryo, LDH-1 is predominant, suggesting that chick
tissues rely more on aerobic metabolism.

Stadtman and coworkers (1961) investigated aspartokinase isoenzyme, in E.

coli. It is responsible for catalyzing the first step of reactions that leads to
formation of lysine, threonine and methionine. They were successful in
separating the aspartokinase activity of whole homogenates into three
fractions. One fraction is found to be inhibited by lysine, another by threonine
and the third by homoserine which is a precursor of methionine.

The isoenzyme cytochrome P450 is involved in degradation and biosynthesis of

endogenous compounds like steroids, fatty acids and cholesterol. It is also
found to perform physiological roles in brain, for example: signal transduction
by arachidonic acid metabolites; the regulation of cerebral vascular tone by
arachidonic acid metabolites, the regulation of corticosteroids and
progesterone in the brain which are believed to affect the mood and state of
excitement; and also control the intracellular concentration of cholesterol. The
pathways modulated by the enzyme P450 enzymes results in formation of
metabolites (1A1, 1A2, 2B1, 2B2, 2C11) that may be responsible in regulating
the ion permeability, enzyme activity and turnover of the membranes. A well-
known polymorphic isoenzyme is debrisoquine hydroxylase enzyme also
known as CYP2D6. The enzyme plays a pivotal role in the metabolism of many
endogenous substances such as neurosteroids, monoaminergic
138 neurotransmitters, neurotransmitters such as serotonin and dopamine and
Unit 9 Multienzyme Complexes
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hence, believed to have a role in determining the psychological state and
behavior of an individual.

9.5.3 Pharmacological Role of Isoenzymes

The study of isoenzymes in serum has been useful in diagnostic procedures

from a very long time. The specificity of information gained from such assays
is dependent upon the tissue distribution of the enzymes. The LDH isoenzyme
in normal serum are present in the proportion of LDH-2 > LDH-1 > LDH-3 >
LDH-4 > LDH-5. Myocardial infractions have suggested changes in serum
levels as compared to normal. There is a marked increase in LDH-1 and
LDH-2 levels. Determination of LDH isoenzyme activity appears to be more
specific and long-lasting indication of tissue damage. Patients with hepatitis
exhibit increase in LDH-5 activity, which is normally present in trace amounts.

The isoenzyme cytochrome P450 in the liver performs important role in

metabolism of drug by transforming them from a hydrophobic state to a much
more easily excretable form. The reaction is grouped into two forms; the
cytochrome P450 dependent phase I and phase II conjugation reactions.
Enzyme inhibition, polymorphism, enzyme induction and various other
physiological factors may lead to alterations in the activity of cytochrome P 450.
These factors have many clinical consequences as they may change the drug
pharmacokinetics leading to its reformed efficacy. This may also result in
amplified toxicity due to reduction in metabolism or increase in the formation of
drug interactions and toxic metabolites.

SAQ 4
Do as Directed

a) Isozymes or Isoenzymes are proteins with ............................... structure

which catalyze the same reaction. (similar/different) [Fill in the blank].

b) The degree of resolution by electrophoresis is generally ineffective than

any other approaches of protein separations. (True/False).

c) From the physiological point of view, isozymes are similar enzymes with
different characteristics, “customized” to specific tissue requirements or
metabolic conditions. (True/False).

d) Arrange in the decreasing order of the proportion of five LDH isoenzymes

present in serum LDH-1, LDH-2, LDH-3, LDH-4, LDH-5.

9.6 SUMMARY
1) Ordered association of various enzymes catalyzing successive steps in a
reaction sequence are called as multienzyme complexes. Several
examples of these complexes include pyruvate dehydrogenase complex,
glycine decarboxylase, tryptophan synthase etc.
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2) There are several advantages of enzymes being physically associated
with one another in an enzyme complex. Transit time is reduced along
with channeling and compartmentation in multienzyme complexes.
3) Instances of coordinate activation of whole multienzyme proteins have
also been observed.
4) Isolation and characterization of multienzyme complexes is reasonably
more complicated than that of a single enzyme. These complexes are
more common in eukaryotes as compared to prokaryotes.
5) Multiple forms of same enzyme are called as isozymes. The distribution of
isozymes of a given enzyme shows different metabolic patterns in
different organs. They may have variable metabolic and regulatory roles.
Lactate dehydrogenase was one of the first enzymes to be identified as
isozyme. The pharmacological role of isoenzymes is extremely useful in
diagnostic procedures. Activity of LDH isoenzyme emerges to be more
definite and long-lasting indication of the tissue damage.

9.7 TERMINAL QUESTIONS

1) What are the characteristic properties of multienzyme complexes?

2) Enumerate the role of pyruvate dehydrogenase multienzyme complex.

3) Distinguish between multienzyme complex and multienzyme

polypeptides.

4) Explain the physiological and pharmacological role of isozyme lactate

dehydrogenase.

9.8 ANSWERS
Self-Assessment Questions
1) a) True, b) Stable, c) False, d) False

2) a) prokaryotes, b) more, c) less, d) Channeling

3) a) True, b) False, c) False, d) True.

4) a) different, b) False c) True, d) LDH-2 > LDH-1 > LDH-3 > LDH-4
> LDH-5.

Terminal Questions
1) Refer to section 9.4

2) Refer to section 9.4.1

3) Refer to section 9.2

4) Refer to section 9.5.1

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9.9 SUGGESTED READINGS

1) David L. Nelson and Michael M. Cox: Lehninger Principles of Biochemistry
6th Ed., W.H. Freeman.

2) Robert K. Murray, Daryl K. Granner, Victor W. Rodwell Harper’s Illustrated

Biochemistry, 27th edition. 2006, McGraw-Hill.

3) Donald J Voet and Judith G. Voet:Principles of Biochemistry4th ed., John

Wiley and Sons, Inc, USA.

4) Eric E Conn, Paul K Stumpf: Outlines of Biochemistry, John Wiley and

Sons, Inc, USA.

5) S. Shanmugan and T. Sathishkumar: Enzyme Technology, I K International

Publishing House Pvt Ltd, New Delhi.

6) Nicholas C Price and Lewis Stevens: Fundamentals of Enzymology,

Oxford University Press, Oxford, New York, USA.

7) Isoenzymes by John Hennery Wiliknson, Springer Science & Business

Media, 06-Dec-2012.

8) Organized Multienzyme Systems: Catalytic Properties by G. Rickey

Welch, Academic Press, 28th January 1985.

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