The document provides an in-depth exploration of multienzyme complexes, highlighting their structural and functional attributes in biochemical pathways. It discusses the significance of these complexes, such as the pyruvate dehydrogenase complex, in catalyzing metabolic reactions and regulating energy production in cells. Additionally, it details mechanisms of enzyme regulation, including the role of various cofactors and the impact of metabolic conditions on enzyme activity.

1. Multienzyme System: Structure & Dynamics
Semester II, Paper CC – 7, Unit II
Dr. Khushbu Kumari
Ph.D, CSIR NET LIFE SCIENCES

2. INTRODUCTION
➢ Multienzyme complex contains several copies of one or several enzymes (polypeptide chains)
packed into one assembly. Multienzyme complex carries out a single or a series of biochemical
reactions taking place in the cells. It allows to segregate certain biochemical pathways into one
place in the cell.
OR
➢ Multienzyme complexes are discrete and stable structures composed of enzymes associated
noncovalently that catalyze two or more sequential steps of a metabolic pathway.
➢ Examples include pyruvate dehydrogenase, fatty acid synthetase, glutamine
synthetase, proteasome, rubisco.
➢ A multienzyme complex that functions in the histidine biosynthesis pathway has been studied at
the biochemical and genetic level in the fungus Neurospora crassa. A gene (His-3) was found to
encode a protein that functions as a multienzyme complex having three distinct enzymatic
activities in the biosynthesis pathway.
➢ A genetic analysis of mutants defective in the N. crassa histidine pathway indicated that the
individual activities of the multienzyme complex occur in discrete regions of the His-3 genetic
map. This finding suggested that each of the activities of the multienzyme complex are encoded
separately from each other, but within the same gene.
➢ Some His-3 mutants were also found that lacked all three activities simultaneously, suggesting
that some mutations can cause loss of function of the whole multienzyme complex.

3. ➢ Many enzymes in living cells catalyse chains of reaction in a sequential order either in a
biosynthetic or a catabolic pathway. In the preceding paragraphs hypothetical
examples of such pathways have been cited. The series of enzymes catalyzing such
chains of reactions are said to form a multi-enzyme system.
➢ In its simplest form, the enzymes of such a system remain free in the cytosol as
independent entities each interacting with its own substrate which is also present in -
the cytosol. The product formed from each reaction is liberated and is acted upon by
another enzyme of the sequence. Some of the multi-enzyme systems may operate in a
different way, when the enzymes are closely associated with each other to form a multi-
enzyme complex.
➢ The fatty acid synthetase of yeast provides an example of a multi-enzyme complex. It
consists of seven different enzymes which form a tightly bound cluster. Each enzyme of
the complex catalyzes a different reaction, ultimately producing a long- chain fatty
acid.
➢ Not only are the enzymes bound in the complex, but also the intermediates of the
synthetic pathway remain bound to it and only the final product is released. Another
example is the respiratory chain enzymes. In eukaryotic cells, these enzymes form the
electron transport system and are physically located in the inner membrane of the
mitochondrion. In the prokaryotic cells, they form part of the cytoplasmic membrane.

5. OCCURRENCE
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.
S1 S2 S3 S4
E1 E2 E3 E4 E5
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.

6. ISOLATION
➢ 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,
dihydroorotase (CAD). Examples include enzymes having kinase and phosphatase activities present in
the same polypeptide is isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase.

7. Pyruvate dehydrogenase complex
✓ Pyruvate dehydrogenase complex (PDC) is a complex of three enzymes that
converts pyruvate into acetyl-CoA by a process called pyruvate decarboxylation.[1] Acetyl-
CoA may then be used in the citric acid cycle to carry out cellular respiration, and this
complex links the glycolysis metabolic pathway to the citric acid cycle. Pyruvate
decarboxylation is also known as the "pyruvate dehydrogenase reaction" because it also
involves the oxidation of pyruvate.[2]
✓ This multi-enzyme complex is related structurally and functionally to the oxoglutarate
dehydrogenase and branched-chain oxo-acid dehydrogenase multi-enzyme complexes.

8. Localization of PDC
➢ In eukaryotic cells the pyruvate decarboxylation occurs inside the mitochondrial
matrix, after transport of the substrate, pyruvate, from the cytosol.
➢ The transport of pyruvate into the mitochondria is via the transport
protein pyruvate translocase.
➢ Pyruvate translocase transports pyruvate in a symport fashion with a proton
across the inner mitochondrial membrane.

9. History
➢ 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.

10. Introduction
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.

11. E.coli pyruvate dehydrogenase multienzyme
complex
✓ Lester Reed and coworkers, in 1968 reported that the E.coli 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 noncovalent 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.

13. ✓ Apart from E.coli, the complex has also been studied in various other
organisms and tissues which include: Bacillus stearothermophilus, Azotobacter
vinelandii, Pseudomonas spp, Saccharomyces cerevisviae, 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.
✓ 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

16. How the activity of this multienzyme complex in
prokaryotes E.coli 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.
✓ 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. 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 Ca2+ and Mg2+which is responsible for the
removal of this phosphate.

19. ✓ 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 E2 has binding
sites for both E1 and E3 but
E1and E3 do not bind when
E2 is absent.

20. Regulation of PDC Complex
✓ Pyruvate dehydrogenase is inhibited when one or more of the three following ratios are
increased: ATP/ADP, NADH/NAD+ and acetyl-CoA/CoA.
✓ In eukaryotes PDC is tightly regulated by its own specific pyruvate dehydrogenase kinase (PDK)
and pyruvate dehydrogenase phosphatase (PDP), deactivating and activating it respectively.[12]
✓ PDK phosphorylates three specific serine residues on E1 with different affinities. Phosphorylation
of any one of them (using ATP) renders E1 (and in consequence the entire complex) inactive.[12]
✓ Dephosphorylation of E1 by PDP reinstates complex activity.[12]
✓ Products of the reaction act as allosteric inhibitors of the PDC, because they activate PDK.
Substrates in turn inhibit PDK, reactivating PDC.
✓ During starvation, PDK increases in amount in most tissues, including skeletal muscle, via
increased gene transcription. Under the same conditions, the amount of PDP decreases. The
resulting inhibition of PDC prevents muscle and other tissues from catabolizing glucose
and gluconeogenesis precursors. Metabolism shifts toward fat utilization, while muscle protein
breakdown to supply gluconeogenesis precursors is minimized, and available glucose is spared for
use by the brain.
✓ Calcium ions have a role in regulation of PDC in muscle tissue, because it activates PDP,
stimulating glycolysis on its release into the cytosol - during muscle contraction. Some products of
these transcriptions release H2 into the muscles. This can cause calcium ions to decay over time.