Enzymology (BT306)

(2 credit)
BT306 (B Sc BT Sem IV)
Unit 1 – Introduction to enzymes
Unit 2: Regulation of Enzyme Activity
Unit 3: Enzyme Technology
Unit 4: Immobilization of enzymes

Jan 3-10, 2024

Unit 2 – Regulation of Enzyme Activity

Week 1 5.1 Enzyme inhibition: types of inhibition, determination of Ki, suicide inhibitor.
5.2 Enzyme regulation: Product inhibition, feedback control, covalent modification.

Week 2 6.1 Allosteric enzymes with special reference to aspartate transcarbomylase and phosphofructokinase.
6.2 Qualitative description of concerted and sequential models. Negative co-operativity and half site
reactivity.

Week 3 7.1 Isoenzymes– multiple forms of enzymes with special reference to lactate dehydrogenase.
7.2 Multienzyme complexes. Ribozymes. Multifunctional enzyme-eg Fatty Acid synthase.
 Enzyme reaction
 Enzyme is a protein (except few RNA) acts as biocatalysts. They catalyze a biochemical reaction by lowering its activation
energy, therefore they can increase the rate of reaction by several folds (104 to 1017 fold)
 As diastase, amylase was the first enzyme to be discovered and isolated by Anselme Payen in 1833.
 Regulation of enzyme activity
 Enzymes are proteins, they are often regulated as any other protein by proteolytic
enzymes or ubiquitination system which degrades protein in to their amino acids.
 As unnecessary activity within a cell can be wasteful or harmful, enzymes/ metabolic
pathways are regulated by four primary means:
1) Proteolytic cleavage (irreversible covalent modification- Zymogen activation),
2) Control proteins (TF which enhances protein expression, binding with Co-enzymes
or proteins to activate or inhibit their activity – Calmodulin and G -protein )
3) Allosteric regulation (metabolic regulators – activators or inhibitors cause change
in enzyme conformation).
4) Reversible covalent modification (phosphorylation/ dephosphorylation,
methylation)
 Allosteric regulation of enzymes
 Allosteric inhibition -Allosteric inhibition is a type of enzyme inhibition where the inhibitor slows down the
enzyme activity by deactivating the enzyme and binding to the enzyme at the allosteric site.
 Here, the inhibitor does not directly compete with the substrate at the active site. But, it indirectly changes the
composition of the enzyme (3D structure). Once the shape is changed, the enzyme becomes inactive. Thus, it can
no longer bind with the corresponding substrate. This, in turn, slows down the formation of final products.

 Allosteric inhibition is a type of enzyme inhibition where the inhibitor

slows down the enzyme activity by deactivating the enzyme and
binding to the enzyme at the allosteric site.
 Here, the inhibitor does not directly compete with the substrate at the
active site. But, it indirectly changes the composition of the enzyme
(3D structure). Once the shape is changed, the enzyme becomes
inactive. Thus, it can no longer bind with the corresponding substrate.
This, in turn, slows down the formation of final products.
 The key difference between non-competitive and allosteric inhibition
is that in non-competitive inhibition, the maximum rate of catalyzed
reaction (Vmax) decreases and substrate concentration (Km) remains
unchanged, while in allosteric inhibition, Vmax remains unchanged
and Km increases.
  Lineweaver-Burk plot (double reciprocal plot) can help us to understand the type of inhibitor or mechanism of
inhibition as follows -

 In non–competitive inhibition, the Vmax of the reaction decreases while leaving the Km value unchanged. In
contrast, in allosteric inhibition, the Vmax remains unchanged, and the Km value increases. So, this is the key
difference between non–competitive and allosteric inhibition
Enzyme regulation in metabolic pathways
 In our body many metabolic pathways are operating all the timein mammals/ Living organisms.
Metabolic pathways consist of series of enzymatic reactions.
 Enzymes which are part of a metabolic pathway need to be regulated in order to control the
metabolic process such rate of glycolysis, gluconeogenesis. Many key enzymes are regulated
directly or indirectly by these metabolic regulators.
 Living organisms need to generate energy continuously to maintain cellular processes and functions.
Same time they don’t waste energy in futile exercise.
 There are many Metabolic regulators (enzyme activity regulators) present in the body or generated
during metabolic reactions which regulate several anabolic or catabolic pathways by regulating key
enzyme activity in the pathway.
 Enzyme regulators can follow different mechanisms such as Product inhibition, Feedback
inhibition, covalent modification and allosteric regulation to self-regulate the metabolic pathway.
 Metabolic regulators of Enzyme
 Metabolic regulators - Metabolic regulation is a term used to describe the process by which metabolic pathways
(both the anabolic/biosynthetic and catabolic/degradative pathways) are regulated in mammals/ Living organisms.
 Living organisms need to generate energy continuously to maintain cellular processes and functions. Same time they
don’t waste energy in futile exercise. There are many Metabolic regulators present in the body or generated during
metabolic reactions which regulate several anabolic or catabolic pathways by regulating key enzyme activity in the
pathway.
 For intrinsic regulation of metabolic pathways the reactions which self-regulate to respond to changes in the levels
of substrates or products. For example, a decrease in the amount of product can increase the metabolic pathway. This
is called a feedback mechanism.
 Metabolic pathways consist of series of enzymatic reactions. Many key enzymes are regulated directly or indirectly by
these metabolic regulators.
 Glycolysis is the process where glucose in coveted into pyruvate by 10 sequential enzymatic reactions.
 Glycolysis is regulated at the steps catalyzed by hexokinase, PFK-1, and pyruvate kinase. Hexokinase is the initial
enzyme of glycolysis, catalyzing the phosphorylation of glucose by ATP to glucose-6-P.
 The pyruvate dehydrogenase complex (PDH) is regulated by covalent modification through the action of a specific
kinase and phosphatase; the kinase and phosphatase are regulated by changes in NADH, acetyl-CoA, pyruvate, and
insulin.
Metabolic regulators of different enzymes -
 The three key enzymes of glycolysis
are hexokinase, phosphofructokinase, and
pyruvate kinase which are allosterically modulated
by various metabolic intermediates or metabolites.

 G-6-P is an allosteric inhibitor of Hexokinase. AMP

and ADP activate HK and ATP inhibit HK activity.

 Increased ATP concentration act as an allosteric

inhibitor of PFK and PK enzymes.

 Increased AMP, ADP and Pi concentration act as

an allosteric activator of PFK and PK enzymes.

 Acetyl Co A and citric acid also inhibit PFK and PK

enzymes.

 F-6-P, F2,6,BisP are a positive regulator of PFK

enzymes enhancing glycolysis process.
 Metabolic regulators of Hexokinase enzyme -
 Hexokinase thus provides a good example of induced fit (allosteric regulation). When glucose is not present, the
enzyme is in an inactive conformation with the active-site amino acid side chains out of position for reaction.

 When glucose (but not water) and Mg+ ATP bind, the binding energy derived
from this interaction induces a conformational change in hexokinase to the
catalytically active form.
 Hexokinase (M 107,862) is a bisubstrate enzyme that catalyzes the reversible
reaction. The enzyme can phosphorylate glucose and water. It can discriminate
between glucose and water because of a conformational change in the enzyme
when the correct substrates binds (fig 6-22).

ATP and ADP always bind to enzymes as a complex

with the metal ion Mg2+.
Metabolic regulators of Hexokinase enzyme -
 This model has been reinforced by kinetic studies.
 The five-carbon sugar xylose, stereo chemically similar to glucose but one carbon shorter, binds to hexokinase
but in a position where it cannot be phosphorylated. Nevertheless, addition of xylose to the reaction mixture
increases the rate of ATP hydrolysis.

 Evidently, the binding of xylose is sufficient to induce a change in hexokinase to its active conformation, and the
enzyme is thereby “tricked” into phosphorylating water.
 The hydroxyl at C-6 of glucose (to which the #- phosphoryl of ATP is transferred in the hexokinase reaction) is similar
in chemical reactivity to water, and water freely enters the enzyme active site. Yet hexokinase favors the reaction
with glucose by a factor of 106.

 The hexokinase reaction illustrates that enzyme specificity is not

always a simple matter of binding of one compound but not
another.
 In the case of hexokinase, substrate specificity is observed not in
the formation of the ES complex but in the relative rates of
subsequent catalytic step.
 Another instance in which negative allosteric modulation can be seen is between ATP and the enzyme
Phosphofructokinase within the negative feedback loop that regulates glycolysis. Phosphofructokinase (generally
referred to as PFK) is an enzyme that catalyses the third step of glycolysis, i.e. the phosphorylation of Fructose-6-
phosphate into Fructose 1,6-bisphosphate.
 PFK can be allosterically inhibited by high levels of ATP within the cell. When ATP levels are high, ATP will bind to an
allosteric site on phosphofructokinase, causing a change in the enzyme's three-dimensional shape. This change causes
its affinity for substrate (fructose-6-phosphate and ATP) at the active site to decrease, and the enzyme is deemed
inactive.
 This causes glycolysis to cease when ATP levels are high, thus conserving the body's glucose and maintaining balanced
levels of cellular ATP. In this way, ATP serves as a negative allosteric modulator for PFK, despite the fact that it is also a
substrate of the enzyme.
 Allosteric regulation - Ligand binding & Cooperative binding
 If more than one ligand-binding site is present on a protein, there is a possibility of interaction between the
binding sites during the binding process.
 Binding of one ligand to its binding site might increase (positive allostery) or decrease the binding affinity of other
ligand binding sites (Negative allostery). is present on a protein, there is a possibility of interaction between the binding
sites during the binding process.
 Ligand binding & Cooperative binding
• The any protein-ligand binding can show co-operativity (positive/ negative or no co-operation). One can
compare the ligand-binding affinity of two molecules/ proteins using Hills equation (Hills plot).

 Hills equation measure the level of cooperativity between

different molecules and their ligands

 In above graph, two proetins (X & Y like Mb and Hb) show different affinity to a common substrate (Oxygen).
 Protein X require less amount of substrate to achieve 50% saturation (50% binding sites are occupied) while protein Y
require more substrate concentration to achieve 50% saturation .
 This indicates protein X has more affinity for the substrate compared to protein Y.
 Ligand binding & Cooperative binding
 Hills equation measure the level of cooperativity between a molecules and their ligands. The Hill coefficient (nH) is a
central parameter in the study of ligand-protein interactions, which measures the degree of cooperativity between
subunits that bind the ligand in multisubunit proteins.

Mb oxygen saturation curve is straight while Hb

oxygen saturation curve is sigmoidal because of
cooperative ligand binding in case of Hb.

• Theta - is the fraction of the protein concentration that is bound by the ligand.
• L is the total ligand concentration,
• Kd is dissociation constant
• KA = affinity constant/ Ligand conc producing 50% saturation.
• n = is the Hill coefficient.
 Allosteric regulators of different enzymes -
 Sigmoidal kinetic profiles are the result of enzymes that demonstrate positive cooperative binding. cooperativity
refers to the observation that binding of the substrate or ligand at one binding site affects the affinity of other sites for
their substrates.
 Negative allosteric modulation (also known as allosteric inhibition) occurs when the binding of one
ligand decreases the affinity for substrate at other active sites. For example, when 2,3-BPG binds to an allosteric site
on hemoglobin, the affinity for oxygen of all subunits decreases. This is when a regulator is absent from the binding
site.
 The degree of cooperativity is determined by Hill equation for non-Michaelis-Menten kinetics. The Hill equation
accounts for allosteric binding at sites other than the active site. n is the "Hill coefficient."
 Hill Equation is used to model biological interactions that
demonstrate sigmoidal response. The Hills equation is used to
capture the biomolecular interaction that exhibit cooperativity
among two binding molecules.
 The Hill coefficient (nH) is a central parameter which measures
the degree of cooperativity between subunits that bind the
ligand in multisubunit proteins.
n = 1 (no cooperativity/ independent binding)

n < 1 (negative cooperativity)

n > 1 (positive cooperativity)
 Allosteric enzymes regulation theories
 In 1963, Monod –Wyman -Changeux put forward the allosteric
theory of regulation. They pointed out that these naturally-occurring
metabolic regulators (also called effectors and modifiers) generally
do not resemble the substrate in structure, so are likely to bind to
the enzyme at a separate site and affect the binding of the substrate
by heterotropic cooperativity.
 The word allosteric was originally used to stress the difference in
shape between regulator and substrate (allo meaning other). Since
then it has been used loosely to describe any kind of cooperative
effect, homotropic as well as heterotropic This is because it is based
on the assumption that, in a particular protein molecule, all of the
protomers must be in the same conformational state: all must be in
the R-form or all in the T-form, no hybrids being found because of
supposed unfavorable interactions between sub-units in different
conformational states.
 Oligomeric enzyme has two conformational states :R (relaxed) and T
(tense, or taut) in each protein molecule, all subunits have the same
conformation (a.k.a. symmetry model)In the absence of ligand, the
two states are called R0 and T0
  Allosteric enzymes regulation - concerted models.
 The two principal models for allosteric enzyme behavior are called the concerted model and the sequential model. In
1965, Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux proposed the concerted model for the behavior of
allosteric proteins in a paper that has become a classic in the biochemical literature.

 The distinguishing feature of this model is that the conformations of all subunits change simultaneously. The MWC model
is sometimes referred to as the symmetrical model.
 In the concerted model, the effects of inhibitors and activators can also be considered in terms of shifting the equilibrium
between the T and R forms of the enzyme.
 In their model it was assumed that an allosteric enzyme (or protein) exists in equilibrium between two symmetric states;
inactive and active (T and R states). The transition between these states was assumed to be concerted, that is the
symmetry of the macromolecular assembly is conserved.

 Furthermore the active state has a greater affinity

for substrate than the inactive state. From these
considerations the activity of the enzyme
depends on the position of the equilibrium
between the inactive and active states.

Concerted model of allostery

 Allosteric enzymes regulation - concerted models
 Thus increasing the substrate concentration drives the equilibrium to the active form and gives rise to a sigmoidal
relationship between the initial velocity of the reaction and the substrate concentration.
 The equilibrium can be altered by allosteric effectors that preferentially bind to either the inactive or active state of
the enzyme. This is the basis of feedback inhibition whereby the product of a biosynthetic pathway inhibits the
enzyme that catalyzes their own production.
 This shifting of the equilibrium is responsible for the observed allosteric effects. The MWC model has been shown
mathematically to explain the sigmoidal effects seen with allosteric enzymes.
 The binding of inhibitors to allosteric
enzymes is cooperative; allosteric inhibitors
bind to and stabilize the T form of the
enzyme.
 The binding of activators to allosteric
enzymes is also cooperative; allosteric
activators bind to and stabilize the R form of
the enzyme.
 Allosteric enzymes regulation - sequential models
 The sequential model considers the induced-fit model of substrate binding whereas the concerted model focuses on
perturbing the equilibrium between the T and R states.
 The Daniel Koshland work is associated with the direct sequential model of allosteric behavior. The distinguishing
feature of this model is that the binding of substrate induces the conformational change from the T form to the R form-
the type of behavior postulated by the induced-fit theory of substrate binding.

 A conformational change from T to R in one subunit makes the same conformational change easier in another
subunit, and this is the form in which cooperative binding is expressed in this model (Figure 7.7a).

 Positive cooperativity can be explained by the

sequential model but not by the concerted model.
 Allosteric enzymes regulation - sequential model
 In the sequential model, the binding of activators and inhibitors also takes place by the induced-fit mechanism.

 The conformational change that begins with binding of inhibitor or activator to one subunit affects the
conformations of other subunits. The net result is to favor the R state when activator is present and to favor the
T form when inhibitor, I, is present (Figure 7.7b).

 The key difference between concerted and sequential

model of allosterism is that in the concerted mode, a
conformational change in one subunit is necessarily
transmitted to all other subunits, whereas in the
sequential model, a conformational change in one subunit
does not induce a similar change in the others.
The MWC equation is consistent with a sigmoidal binding
curve, even though its derivation assumes that the binding of
one molecule of ligand does not affect the affinity for the
ligand of other binding sites on the molecule. The explanation
for the cooperative effects lies in the Rn/Tn equilibrium.

 Sequential model explains Positive cooperativity in better way

than the concerted model.
 Co-Operativity and half site reactivity
 Cooperativity is a feature many multimeric proteins use to control activity. Cooperativity is a fundamental feature of
many multi-subunit enzymes
 Cooperativity critically allows enzymes to make step-like rate responses to substrate concentration changes, and so
offer rapid responses to changing cellular conditions.

 Positive cooperativity implies allosteric binding – binding of the ligand at

one site increases the enzyme's affinity for another ligand at a site different
from the other site. Enzymes that demonstrate cooperativity are defined as
allosteric.

 Negative cooperativity is a phenomenon in which the binding of a first

ligand or substrate molecule decreases the rate of subsequent binding. This
definition is not exclusive to ligand-receptor binding, it holds whenever two
or more molecules undergo two successive binding events.

 Negative cooperativity and positive cooperativity can make a multimeric

enzyme’s response more graded towards substrate binding than it would
otherwise.
 It is not uncommon for proteins to display both positive and negative
cooperativity to the same ligand.
Negative co-operativity
 Negative cooperativity is a phenomenon in which the binding of one or more molecules of a ligand to a multimeric
receptor makes it more difficult for subsequent ligand molecules to bind.
 Glyceraldehyde-3-phosphate dehydrogenase (yeast, muscle, sperm) has long been studied as a model of cooperativity.
Depending on the source, G3PDH can exhibit either positive (sperm enzyme) or negative cooperativity (somatic enzyme)
in coenzyme NAD+ binding. Similarly many DNA binding proteins (TF) shows both positive and negative cooperativity.

 GAPDH is ubiquitously expressed and is found in all domains of life (key

glycolytic enzyme). It is a tetrameric protein and one subunit is 37 kDa.
GAPDH has been implicated in many diseases, including those of pathogenic,
cardiovascular, degenerative, diabetic, and tumorigenic origins.
 Example of Negative cooperativity in enzyme is the binding of
diphosphopyridine nucleotide (NAD) to muscle glyceraldehyde-3-phosphate
dehydrogenase (G3PDH).
 The binding of the coenzymes NAD+ and nicotinamide-1-N6-ethenoadenine
dinucleotide (NAD+ analogues) to rabbit muscle apo-glyceraldehyde-3-
phosphate dehydrogenase exhibits strong negative cooperativity, whereas
acetylpyridine adenine dinucleotide, ATP, and ADP-ribose bind
noncooperatively to the NAD+ sites.
 Interdomain salt bridge D311–H124 is specific for GAPDS. Disruption of the salt bridge D311–H124 eliminates cooperative
NAD+ binding. Disruption of the salt bridge D311–H124 enhances enzymatic activity twofold.
 GAPDH has been shown to possess many key functions in cells. These functions are regulated by protein oligomerization,
biomolecular interactions, post-translational modifications, and variations in subcellular localization.
 Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) exist as tetramer (37KDa of each subunit) which contain single
thiol group (Cys149 -SH) which is important for its catalytic activity.
 RNS (free radicals) reacts with the Cys 149 in the active site, which promote binding of NADH and inhibit G3PDH enzyme
activity. This results decrease in glycolysis and this is the cytotoxic mechanism of RNS also.
 3-Br-isoxazoline is a potent antimalarial drug which fully inhibit glyceraldehyde 3-phosphate dehydrogenase from
plasmodium falciparum by selectively alkylating all four catalytic cysteines of the tetramer.

 Under the same experimental conditions that led to a fast and

complete inhibition of the protozoan enzyme, the human
ortholog was only 25% inhibited, with the alkylation of a single
catalytic cysteine within the tetramer.
 The partial alkylation seems to produce a slow conformational
rearrangement that severely limits the accessibility of the
remaining active sites to bulky 3-Br-isoxazoline derivatives, but
not to the substrate or smaller alkylating agents.
 Half site reactivity
 Negative cooperativity is a phenomenon in which the binding of a first ligand or substrate molecule decreases the
rate of subsequent binding. The extreme form of negative homotropic cooperativity is the so-called “half-site”
reactivity, where enzyme active sites are paired, and only one can be active at a time.
 Many enzymes consisting of tetramers or higher order multimers are made from two or more pairs of such coupled
sites. All the subunits do not perform symmetrically.
 G3PDH is a tetramer enzyme but it exist as dimer of dimer. Negative cooperativity in NAD binding to wild-type GAPDH
is interpreted according to the induced-fit model in terms of two independent dimers with two interacting binding
sites in each dimer.
 The results are consistent with a built-in asymmetry of the tetramer and suggest that the arginine residue (probably
Arg-231) controls the conformational transition between the asymmetric and symmetric states of the tetramer.

 Modification of a single arginine residue per

subunit of rabbit muscle apo-D-
Glyceraldehyde-3-phosphate dehydrogenase
does not affect the rate of hydrolysis of p-
nitrophenyl acetate catalyzed by the enzyme,
but locks the tetramer in a conformation
wherein only two active sites are functioning
(negative co-operativity and half site
allostery).
 Half site reactivity
 Interdomain salt bridge D311–H124 is specific for GAPDS. Disruption of the salt bridge D311–H124 eliminates
cooperative NAD+ binding.
 Two dimeric mutant GAPDHs, i.e. Y46G/S48G and D186G/E276G, were shown to exhibit positive cooperativity in NAD
binding. Such an effect was caused by the disruption of the interdomain salt bridge D311–H124, which is located close
to the active site of the enzyme.
 The modified enzyme also exhibits half-of-the sites reactivity towards iodoacetate and iodoacetamide. On the other
hand, its NAD+-binding characteristics remain unchanged.
 Half site reactivity
 Cytidine-5′-triphosphate (CTP) synthase (CTPS) is the class I glutamine-dependent amidotransferase (GAT) that catalyzes
the last step in the de novo biosynthesis of CTP. Glutamine hydrolysis is catalyzed in the GAT domain and the liberated
ammonia is transferred via an intramolecular tunnel to the synthase domain where the ATP-dependent amination of UTP
occurs to form CTP.
 CTPs catalyzes the last step of CTP biosynthesis, where nascent ammonia generated at the glutaminase site reacts with the
ATP-phosphorylated UTP to produce CTP at the synthetase domain. Since CTPs is essential for RNA, DNA, and phospholipid
biosynthesis, its biochemistry and kinetics have been extensively studied.

 CTP synthase enzyme is a recognized target for the development of anticancer,

antiviral, and antiprotozoal agents.
 Half site reactivity
 It has recently been shown that the active enzyme is a tetramer of molecular weight 200,000. CTPS1 is an asymmetrical
homotetramer with only three of its four monomers contributing to the catalytic domain.
 CTPS is unique among the glutamine-dependent amidotransferases, requiring an allosteric effector (GTP) to activate the
GAT domain for efficient glutamine hydrolysis.

 The addition of an allosteric effector GTP induces a further conformational change to stimulate glutamine hydrolysis.
Therefore, CTPs possesses individual recognition sites for glutamine, UTP, ATP, and GTP.
 The enzyme exhibits positive homotropic effects toward ATP and UTP. The ATP and UTP binding domains are located at
the tetramer interface, whereas the glutamine binding domain is located away from the tetramer interface.
 In the examination of the kinetic properties of this enzyme, negative cooperative effects (half site reactivity).
 Guanosine triphosphate (GTP) is an allosteric activator of enzyme activity which stimulates the hydrolysis of glutamine.
 CTP is an allosteric inhibitor of enzyme activity; the CTP binding site overlaps with and impedes the UTP binding site.
Thus, CTPS1 enzymatic activity is sensitive to the levels of all four essential ribonucleotides.
Allosteric enzymes regulation –
1. Phosphofructokinase.
 Allosteric enzymes regulation - Phosphofructokinase.
 Glycolysis is the foundation for respiration, both anaerobic and aerobic. Because phosphofructokinase (PFK) catalyzes the
ATP-dependent phosphorylation to convert fructose-6-phosphate into Fructose 1, 6, bisphosphate, it is one of the key
regulatory and rate limiting steps of glycolysis.
 Phosphofructokinase-1 (PFK-1) is one of the most important regulatory enzymes (EC 2.7.1.11) of glycolysis. It catalyzes
3rd step of glycolysis.
 PFK-1 catalyzes the important "committed" step of glycolysis, the
conversion of fructose- 6-phosphate and ATP to fructose 1, 6-
bisphosphate and ADP.

 It is an allosteric enzyme made of 4 subunits and controlled by many

activators and inhibitors.
 Negative allosteric modulation can be seen is between ATP and the
enzyme Phosphofructokinase within the negative feedback loop that
regulates the rate of glycolysis.
  The enzyme has two sites with different affinities for ATP which is both a substrate and an inhibitor.

 PFK can be allosterically inhibited by high levels of ATP within the

cell. When ATP levels are high, ATP will bind to an allosteric site on
phosphofructokinase, causing a change in the enzyme's three-
dimensional shape. This change causes its affinity for substrate
(fructose-6-phosphate and ATP) at the active site to decrease, and
the enzyme is deemed inactive.
 This causes glycolysis to cease when ATP levels are high, thus
conserving the body's glucose and maintaining balanced levels of
cellular ATP. In this way, ATP serves as a negative allosteric modulator
for PFK, despite the fact that it is also a substrate of the enzyme.

 PFK is able to regulate glycolysis through allosteric inhibition, and in this way, the cell can increase or decrease the rate
of glycolysis in response to the cell’s energy requirements. For example, a high ratio of ATP to ADP will inhibit PFK and
glycolysis. The key difference between the regulation of PFK in eukaryotes and prokaryotes is that in eukaryotes PFK is
activated by fructose 2, 6-bisphosphate.
 The purpose of fructose 2, 6-bisphosphate is to supersede ATP inhibition, thus allowing eukaryotes to have greater
sensitivity to regulation by hormones like glucagon and insulin.
 Allosteric enzymes regulation - Phosphofructokinase.
 PFK-1 catalyzes the conversion of fructose 6-phosphate and ATP to fructose 1,6-bisphosphate and ADP. PFK-2 catalyzes
the synthesis of fructose 2,6-phosphate from fructose 6-phosphate.
 PFK-1 activity is affected by the ATP concentration. In contrast, PFK-2 is not affected by ATP concentration. PFK-2 is a
bifunctional enzyme, also having the capability of a F2,6-BP phosphatase, and hence of controlling both synthesis and
degradation of F2,6-BP.
 PFK 1 is able to regulate glycolysis through allosteric regulation, and in this way, the cell can increase or decrease the
rate of glycolysis in response to the cell's energy requirements.
 For example, a high ratio of ATP to ADP will inhibit PFK and glycolysis. PFK1 is allosterically inhibited by high levels of
ATP but AMP reverses the inhibitory action of ATP. Therefore, the activity of the enzyme increases when the cellular
ATP/AMP ratio is lowered. Glycolysis is thus stimulated when energy charge falls.

 PFK1 is allosterically inhibited by ATP and citrate (from

the citric acid cycle) and its product. It is also inhibited
by low pH to prevent the accumulation of hydrogen
ions in muscle.
 Presence of Fructose 2, 6 Bisphosphate increases the
PFK1 activity by increasing its affinity for the substrate
(Frucotse-6-phosphate).
 PFK-2 is itself controlled by several mechanisms: the
enzyme binds avidly to ATP and is equally sensitive to
allosteric inhibition by citrate, as is demonstrated by the
inverse relation between citrate and F2,6-BP
concentrations.
Allosteric enzymes regulation –
2. Aspartate transcarbomylase (ACTase)
 Allosteric enzymes regulation - Aspartate transcarbomylase (ACTase)
 Aspartate transcarbomylase (ACTase) synthesizes first step of pyrimidine nucleotide biosynthesis step. Pyrimidine
nucleotides are required for nucleic acid (DNA, RNA), energy storage (ATP, GTP) & enzyme co-factor (NAD, NADP).

 The enzyme aspartate transcarbamoylase (ATCase, EC 2.1. 3.2 of Escherichia coli), which catalyzes the committed step
of pyrimidine biosynthesis, is allosterically regulated by all four ribonucleoside triphosphates (NTPs) in a nonlinear
manner.

 The binding of substrates

to ATCase is ordered with
CP binding before Asp.
Domain closure is
triggered by the ordered
binding of the substrates.

 In the E. coli enzyme, domain closure induces a dramatic quaternary structural change, from the tense
(T) to the relaxed (R) structure, involving an elongation of the molecule by 11 Å as well as rotations of
the catalytic and regulatory subunits
 Allosteric enzymes regulation - Aspartate transcarbomylase (ACTase)
 Aspartate Transcarbamoylase (ATCase) is an allosterically regulated enzyme with unique quaternary structure involving
separable catalytic and regulatory subunits. This allosteric regulation affects the kinetics of the enzyme.
 The Prokarytic ATCase enzyme is an archetypal example of allosteric modulation of fine control of metabolic enzyme
reactions. However, mammalian aspartate transcarbamoylase is not regulated in this manner.

 Escherichia coli aspartate transcarbamoylase (ATCase)

is the textbook example of an enzyme that regulates a
metabolic pathway—namely, pyrimidine nucleotide
biosynthesis—by feedback control and by the
cooperative binding of the substrate, L-aspartate.
 ATCase does not follow Michaelis–Menten kinetics.
Instead, it lies between its low-activity, low-affinity
"tense" (Inactive form) and its high-activity, high-
affinity "relaxed" states (active form).
 ATCase is a textbook example of a molecule under allosteric regulation in which the binding of substrate to one
active site in a molecule increases the likelihood that the enzyme will bind more substrate, a phenomena called
positive cooperativity.
 Negative co-operativity and half site reactivity
 Aspartate transcarbamoylase (ATCase) catalyzes the first committed step pyrimidine biosynthesis. It shows – -
positive homotropic cooperativity with aspartate (Substrate)
- Negative heterotropic cooperativity with CTP (End product inhibition)
- Positive heterotropic cooperativity with ATP PALA (N-(phosphonacetyl)-L-aspartate) is a bisubstrate
analog of ATCase (i.e contains parts that mimic both substrates aspartate and carbamoyl phosphate).
 Low concentrations PALA activate ATCase but high concentrations of PALA inhibit ATCase.
 Using the concerted two-state model of allostery (i.e the Monod-
Wyman- Changeux model), ATCase display both negative
heterotropic cooperativity with CTP and positive heterotropic
cooperativity with ATP.
Allosteric enzymes regulation - Aspartate transcarbomylase (ACTase)
 ATP is an allosteric activator of ATCase,
able to increase the activity of the
enzyme by 180% at a 2 mM
concentration. ATP T
 ATP does not affect the maximal rate of
the enzyme (Vmax); instead, it induces
a shift from the inactive T state to the
active R state.
 Allosteric enzymes regulation - Aspartate transcarbomylase (ACTase)
 In prokaryotic cells aspartate transcarbamoylase, an allosteric
protein, is inhibited by the end product CTP and UTP and
activated by ATP.
 Cytidine triphosphate (CTP) is an allosteric inhibitor representing
a classic case of feedback inhibition whereby the end product of
a biosynthetic pathway inhibits an enzyme catalyzing a reaction at
the beginning of the pathway.