Enzyme Regulation

Enzymes

Enzymes are the most remarkable and highly specialized proteins, having extraordinary catalytic
power. They are the reaction catalysts of biological systems and have a high degree of specificity for their
substrates. Enzymes accelerate chemical reactions tremendously and function in aqueous solutions under
very mild conditions of temperature and pH. Enzymes adopt a specific three-dimensional structure, and may
employ organic (e.g. biotin) and inorganic (e.g. magnesium ion) cofactors to assist in catalysis. Enzymes have
molecular weight ranging from about 12,000 to more than 1 million. Some enzymes require no chemical
groups for activity other than their amino acid residues, whereas some enzymes require both a coenzyme
and one or more metal ions for activity. A coenzyme or metal ion which is very tightly associated or covalently
bound to the enzyme protein is called a prosthetic group. A complete catalytically active enzyme together
with its bound coenzyme or metal ions is called a holoenzyme. The protein part of a holoenzyme is known
as apoenzyme. A few RNA molecules called ribozymes also catalyze reactions, with an important example
being some parts of the ribosome. Synthetic molecules called artificial enzymes also display enzyme-like
catalysis.

In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted
into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in
order to occur at rates sufficient for life. Since enzymes are selective for their substrates and speed up only
a few reactions from among many possibilities, the set of enzymes made in a cell determines which
metabolic pathways occur in that cell. Like all catalysts, enzymes work by lowering the activation energy for
a reaction, thus dramatically increasing the rate of the reaction. As a result, products are formed faster and
reactions reach their equilibrium state more rapidly. Inhibitors are molecules that decrease enzyme activity;
activators are molecules that increase activity. Many drugs and poisons are enzyme inhibitors. Activity is also
affected by temperature, pressure, chemical environment (e.g., pH), and the concentration of substrate.

The majority of enzymes contains one domain (simple enzymes), while many are composed of two or
more domains (allosteric enzymes and multi-functional proteins). Most enzymes are designed to function at
a constant rate, but allosteric enzymes are sensitive to physiological controls, and thereby adjust their rate
and determine the flux through the metabolic pathway that they control.

Regulatory enzymes

In the cell, enzymes often work together in groups. These sets of reactions are known as metabolic
pathways. Enormous amount of energy and resources are dedicated for each pathway to carry out different
metabolic functions,thus cells have to regulate the activities of the enzyme very precisely. The activities of
metabolic pathways in cells are regulated by control of the activities of certain enzymes. In such enzyme
systems, the reaction product of one enzyme becomes the substrate of the next. In each metabolic pathway,
some enzymes have a greater effect on the rate of the overall sequence. These regulatory enzymes exhibit
increased or decreased catalytic activity in response to certain signals. In most multienzyme systems, the
first enzyme of the sequence is a regulatory enzyme. Other enzymes in the sequence are usually present at
levels that provide an excess of catalytic activity; they can promote their reactions as fast as their substrates
are made available from preceding reactions.

Enzyme regulation will allow the changing needs of the cell to meet its energy and resource demands. If
a product is available in excess, it could then divert the resources to other needy reactions. If a product is in
demand, it could activate the pathway to produce more of the biomolecule that is needed. Thus regulation
is the process, by which cells can turn on, turn off, or modulate the activities of various metabolic
pathways. The four kinds of enzyme regulation are

• Allosteric regulation
• Reversible covalent modification
• Proteolytic activation or irreversible covalent modification
 • Stimulation and inhibition by control proteins
Allosteric regulation

Allosteric regulation is the regulation of an enzyme or other protein by binding an effector molecule at the
protein's allosteric site (that is, a site other than the protein's active site). Details about allosteric enzymes
and allosteric regulation are given below.

Allosteric enzymes

Enzymes that are able to be regulated by binding specific ligands are defined as allosteric. They have the
ability to change between two or more structural shapes that vary in their ability to bind a substrate, or in
their ability to position a critical catalytic side chain, and therefore in their rate of catalysis. There are two
major groups of allosteric enzymes. One group is regulated by changing their affinity for one substrate, while
keeping their maximum rate fairly constant also known as K-type enzymes. The second group does not
demonstrate significant changes in affinity, but has large changes in the maximum rate, known as V-type
enzymes. Allosteric enzymes function through reversible, non-covalent binding of regulatory
compounds called allosteric modulators or allosteric effectors, which are generally small metabolites or
cofactors, whereas other enzymes are regulated by reversible covalent modification. Some enzymes are
stimulated or inhibited when they are bound by separate regulatory proteins. It is by changing their rate,
faster or slower, in response to changing concentrations of the specific cellular metabolites that allosteric
enzymes recognize and bind that enables such enzymes to be sensitive to some metabolic aspect of the cell.
Since they respond by appropriately altering their activity, allosteric regulatory enzymes act as pacemakers
for their pathway. They are regulatory, since they regulate the pathway in which they function, and also
because they are themselves regulated by the binding of physiological effectors.

Allosteric enzymes undergo conformational changes in response to modulator binding. The modulators
for allosteric enzymes may be stimulatory or inhibitory. Regulatory enzymes for which substrate and
modulator are identical are known as homotropic enzymes. Heterotropic enzymes have substrate other
than the modulator. Allosteric enzymes are generally larger and more complex than non-allosteric enzymes.
Their properties are significantly different from those of simple non-regulatory enzymes. Allosteric enzymes
have one or more regulatory sites other than the active site for binding with the modulator. As the active
site in an enzyme is specific for its substrate, similarly, each regulatory site is specific for its
modulator. Homotropic enzymes have the active site which is same as the regulatory site.

Feedback inhibition by allosteric enzyme

Many of the enzyme-catalyzed reactions that occur in a cell, such as those involved in the biosynthesis of an
amino acid, are carried out in a specific sequence called a biochemical pathway. In such pathways, the
product of one reaction becomes the substrate for the next reaction. If the end product of a pathway, such
as an amino acid, becomes available in the environment, it is unnecessary and wasteful for the cells to
continue to produce the product. Cells therefore have the ability to shut down a pathway when it is not
needed. The reaction occurs at a site on the enzyme that is different from the active site, called the allosteric
site. When the product binds to the allosteric site, the enzyme undergoes a conformational change and can
no longer reacts with its substrate.
When the regulatory enzyme reaction is slowed,
all subsequent enzymes operate at reduced rates
as their substrates are depleted. There is no
substrate for subsequent steps in the pathway
and the final product is no longer synthesized. The
rate of production of the pathway's end product is
thereby brought into balance with the cell's
needs. After the product has been utilized or
broken down and its concentration thus
decreased, the inhibition is relaxed, and the
formation of the product resumes. Such enzymes,
whose ability to catalyze a reaction depends upon
molecules other than their substrates (the ones
upon which they act to form a product),
are said to be under allosteric control. This type of regulation is known as feedback inhibition (figure-1), in
which, the end product of the pathway reacts with the first enzyme, that is unique to the pathway.

One of the first known examples of allosteric

feedback inhibition was the bacterial enzyme system
that catalyzes the conversion of L-thereonine to L-
isoleucine in five steps. In this system, enzyme-1
catalyzes the reaction, which involves the removal of
the amino group from L-thereonine, is called L-
thereonine deaminase (figure-2). This enzyme is
strongly inhibited by the ultimate product of the five
reactions, L-isoleucine. L-isoleucine is quite specific as
an inhibitor; other amino acids or related compounds
do not inhibit that enzyme. In this way the cell
regulates the amount of isoleucine produced. When
the concentration of isoleucine begins to get high, the
whole chain of reactions is shut down by the inhibition
of the first reaction in the series.

Figure 2: The conversion of L-thereonine to L-isoleucine by feedback inhibition

Properties of allosteric enzymes

Allosteric enzymes are unique compared to other enzymes because of its ability to adapt various conditions
in the environment due to its special properties. The special property of Allosteric enzymes is that it contains
an allosteric site on top of its active site which binds the substrate. Another important property of allosteric
enzymes is that it also contains many polypeptide chains with multiple active and allosteric sites.

Aspartate transcarbamoylase (ATcase) is an allosteric enzyme which has 12 polypeptide chains organized
into catalytic and regulatory subunits. The enzyme catalyzes the first step in the synthesis of pyrimidines.
The enzyme functions to catalyze the condensation of aspartate and carbamoyl phosphate to form N-
carbamoylaspartate and orthophosphate. The enzyme ultimately catalyzes the reaction that will yield
cytidine triphosphate (CTP). This allosteric enzyme is unique in that for high products of the final product
CTP, the enzyme activity is low. However, for low concentrations of the final product CTP, the enzymatic
activity is high. The allosteric nature is thus represented as the CTP molecule has a odd configuration or
shape that is unlike the substrates. Rather than binding to the active site, CTP binds to the allosteric site.
Thus, CTP functions as an allosteric inhibitor decreasing the enzymatic activity of the enzyme. When CTP
concentrations remain high and cells in the body need more enzyme, a different allosteric molecule ATP
functions to attach to the allosteric site and functions as enzyme activator enhancing the activity of the
enzyme. Thus, even with high concentrations of CTP, the enzyme activity could be enhanced because of ATP,
which also acts on the allosteric site. This example explains the benefits of allosteric control and the ability
allosteric enzymes to adapt to various conditions of the environment.

The second allosteric enzyme being studied is fructose 1,6-bisphosphatase (FBPase), an enzyme that is
critically involved in the control of gluconeogenesis and is a target for anti-diabetic drugs. In diabetes,
fructose 1,6-bisphosphatase fails to regulate gluconeogenesis resulting in altered blood sugar levels. FBPase
is a homotetramer catalyzing the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate. FBPase is
allosterically inhibited by AMP and fructose 2,6-bisphosphate, however, as opposed to ATCase the catalytic
and regulatory binding sites are on same polypeptide chain. For FBPase the R to T conformational change
involves a rotation of the two dimeric halves of the molecule with respect to each other by about 17°. These
quaternary changes are accompanied by alterations in the tertiary structure, the most significant of which
involve large movements of loops. The allosteric transition from the T to the R state results in the formation
of the catalytically competent active site mainly due to the rearrangement of a loop that brings a metal
binding site in close proximity to the active site.
There are distinct properties of Allosteric Enzymes that makes it different compared to other enzymes.

1. Allosteric enzymes do not follow the Michaelis-Menten Kinetics. This is because allosteric enzymes
have multiple active sites. These multiple active sites exhibit the property of cooperativity, where
the binding of one active site affects the affinity of other active sites on the enzyme.
2. Allosteric Enzymes are influenced by substrate concentration. There are two conformational forms
of allosteric enzymes, i.e., T(tight/tensed or taut) and R(relaxed) forms, which exist in equilibrium.
Modulators and substrates can bind to the R form of the enzyme, whereas the inhibitors can bind
to the T form only (figure-3). At high concentrations of substrate, more enzymes are found in the R
state. When there is insufficient amount of substrate, the enzymes are found in the T state. So the
T and R state equilibrium depends on the concentration of the substrate.
3. Allosteric Enzymes are regulated by other molecules. This is seen when the molecules 2,3-BPG (2,3-
bisphosphoglycerate), and CO2 modulates the binding affinity of hemoglobin to oxygen. 2,3-BPG
reduces binding affinity of O2 to hemoglobin by stabilizing the T- state. Lowering the pH from
physiological pH 7.4 to 7.2 (pH in the muscles and tissues) favors the release of O2. Hemoglobin is
more likely to release oxygen in CO2 rich areas in the body.

Figure 3: Equilibrium between the T form and the R form of allosteric enzyme

Michaelis- Menten kinetics

The primary function of enzymes is to enhance rates of reactions so that they are compatible with the
needs of the organism. To know about functioning of enzymes, a kinetic description of their activity is
needed. The study of chemical reactions catalyzed by enzymes is known as enzyme kinetics.

As enzyme-catalysed reactions are saturable, their rate of catalysis does not show a linear response to
increasing substrate. If the initial rate of the reaction is measured over a range of substrate concentrations
(denoted as [S]), the reaction rate (V0) increases as [S] increases, as shown in figure-4.
(V0)

[S]
Figure 4: Saturation curve for an enzyme which obeys Michaelis-Menten kinetics
 However, as [S] gets higher, the enzyme becomes saturated with substrate and the rate reaches Vmax,
the enzyme's maximum rate. In 1913, Leonor Michaelis and Maud Menten proposed a simple model to
account for these kinetic characteristics. In this model, a specific ES complex is a necessary intermediate in
catalysis. Consider an enzyme that catalyzes the S to P by the following pathway:

An enzyme E combines with substrate S to form an ES complex, with a rate constant k1. The ES complex
can dissociate to E and S with a rate constant k-1, or it can proceed to form product P, with a rate constant
k2. It can also be assumed that almost none of the product reverts to the initial substrate, a condition that
holds in the initial stage of a reaction before the concentration of product is appreciable.

The catalytic rate is equal to the product of the concentration of the ES complex and k2.
V0 = k2 [ES]------------- (1)

The rate of formation of ES is given by:

[ES] = k1 [E] [S]----------- (2)

The rate of breakdown of ES is given by:

[ES] = (k-1 + k2) [ES]------------ (3)

Considering a steady state, [ES], stay the same even if the concentrations of starting materials and
products are changing. This occurs when the rates of formation and breakdown of the ES complex are
equal. Therefore equating equations 2 and 3,

k1 [E] [S] = (k-1 + k2) [ES], which can be modified to

( [E] [S] ) / [ES] = ( k-1 + k2) / k1------------- (4)

Equation 4 can be simplified by defining a new constant, KM, called the Michaelis constant:
KM = ( k-1 + k2) / k1---------------------- (5)

KM is an important characteristic of enzyme-substrate interactions and is independent of enzyme and

substrate concentrations.

Considering equations 4 and 5, [ES] can be determined, which isà

[ES] = ( [E] [S] ) / KM ------------------------- (6)

The concentration of uncombined substrate [S] is very nearly equal to the total substrate concentration,
provided that the concentration of enzyme is much lower than that of substrate. The concentration of
uncombined enzyme [E] is equal to the total enzyme concentration [E]T minus the concentration of the ES
complex. Therefore
[E] = [E]T ─ [ES] ------------------------- (7)

Equation 6 can be written as

[ES] = ( ( [E]T ─ [ES] ) [S] ) / KM ------------------------- (8)
[ES]KM = [E] T [S] ─ [ES] [S]
[ES]KM + [ES] [S] = [E] T [S]
[ES] (KM + [S]) = [E] T [S]
[ES] = ([E] T [S])/ (KM + [S]) ------------------------- (9)
 Substituting the value of [ES] from equation in equation 1,
V0 = k2 [E]T ( [S] / [S] + KM ) ------------------ (10)

The maximal rate, Vmax, is attained when the catalytic sites on the enzyme are saturated with substrate,
that means when
[ES] = [E]T.

Therefore,
Vmax = k2 [E]T -------------------------- (11)

Putting the value of Vmax from equation 11 in equation 10, we get

V0 = Vmax ( [S] / [S] + KM ) ------------------- The Michaelis Menten equation.

The equation states that, at very low substrate concentration, when [S] is much less than KM,
Then,V0 = (Vmax / KM) [S]; that is, the rate is directly proportional to the substrate concentration. At high
substrate concentration, when [S] is much greater than KM, Then V0 = Vmax ; that is, the rate is maximal,
independent of substrate concentration.

Deviation of allosteric enzymes from Michaelis-Menten kinetics

The relationship between V0 and [S] in allosteric enzymes is different from that of Michaelis-Menten kinetics.
They do exhibit saturation with the substrate when [S] is sufficiently high, but for some allosteric enzymes,
plots of V0 verses [S] produce a sigmoid saturation curve (figure-5) rather than the hyperbolic curve of non-
regulatory enzymes (figure-4). In allosteric enzymes, the binding of substrate to one active site can affect
the properties of other active sites in the same enzyme molecule. The binding of substrate to one active site
of the enzyme facilitates substrate binding to the other active sites, and this cooperativity results in a
sigmoidal plot of V0 versus [S]. On the sigmoid saturation curve, the value of [S], at half maximal V0 can't be
designated as KM, because the enzyme doesn't follow the hyperbolic Michaelis-Menten relationship. In the
sigmoidal plot, the substrate concentration at half maximal velocity is designated as [S]0. 5 or K0. 5 .
V0 (µM/min)

[S] (mM)

Figure 5: The sigmoid curve for a homotropic allosteric enzyme

Homotropic allosteric enzymes generally are multi-subunit proteins and the same binding site on each
subunit functions as both the active site and the regulatory site. The substrate acts as a positive modulator
as binding of one molecule of substrate to one binding site alters the enzyme's conformation and enhances
the binding of subsequent substrate molecules. So this results in the sigmoid change in V0, with increasing
substrate concentration. Sigmoid kinetics involves small changes in the concentration of a modulator can be
associated with large changes in activity. The sigmoid curve for a homotropic enzyme (figure-5), shows a
relatively small increase in the substrate concentration, [S] causes a comparatively large increase in V0.
 In heterotropic allosteric enzymes, the modulators are metabolites other than the normal substrate,
and therefore the substrate-saturation curve changes according to the modulator. Some heterotropic
allosteric enzymes have activating modulators, while some have inhibitory modulators and some have
both inhibitory and activating modulators. An activator or positive modulator may cause the curve to
become more nearly hyperbolic, with a decrease in K0. 5 but no change in Vmax, resulting in an increased
reaction velocity at a fixed substrate concentration (Figure-6, upper curve). A negative modulator or an
inhibitor may produce a more sigmoid substrate-saturation curve (Figure-6, lower curve), with an increase
in K0. 5.

+
K0.5 K0.5 -
K0.5

Figure 6: The effects of a positive modulator and a negative modulator on a heterotropic allosteric enzyme

Models of allosteric regulation

The sigmoidal dependence of V0 on [S] reflects subunit co-operativity, has inspired two models to explain
these cooperative interactions. Most allosteric effects can be explained by the concerted MWC model put
forth by Monod, Wyman, and Changeux, or by the sequential model described by Koshland, Nemethy, and
Filmer. Both postulate that enzyme subunits exist in one of two conformations, tensed (T) or relaxed (R),
and that relaxed subunits bind substrate more readily than those in the tense state. The two models
(figure-9) differ most in their assumptions about subunit interaction and the pre-existence of both states.

The concerted model

The concerted model of allostery, postulates that enzyme subunits are connected in such a way that a
conformational change in one subunit is necessarily conferred to all other subunits or in other words, all
subunits must be in the same conformation. As mentioned earlier that allosteric enzymes can exist in two
states, i.e., relaxed (R state) and tight (T state). In this two-state model, all the subunits of an oligomer
must be in the same state (they all change together) and is therefore termed the concerted model . T state
predominates in the absence of substrate S and S bind more tighter to R than T. The T and R state
equilibrium depends upon the concentration of the substrate. At high substrate concentration, more
enzymes are found in the R state, whereas at low substrate concentration, the enzymes are found in the T
state. The equilibrium can be shifted to the R or T state through the binding of the allosteric effector
(activator or inhibitor) to the allosteric site. Activator and inhibitor bind to R and T states respectively
(Figure-8). In this symmetry model, binding of ligand to one subunit always assists the binding of the same
ligand to the next subunit, i.e, only positive co-operativity is possible here. Heterotropic interactions could
either be positive or negative.
 Figure 8: The concerted model for allosteric regulation

The sequential model for allosteric regulation

The sequential model (figure-9) of allosteric regulation holds

that subunits are not connected in such a way that a
conformational change in one subunit induces a similar change
in the other subunits. Thus, all enzyme subunits do not
necessitate the same conformation. Subunits may undergo
individual sequential changes in conformation. Subunits can
interact in different conformations. The sequential model says
that molecules of substrate bind via an induced-fit hypothesis.
When a subunit randomly collides with a molecule of substrate,
the active site, in essence, forms a glove around its substrate.
While such an induced fit of the substrate and a subunit, cause a
conformation change (Figure-9) in the subunit, converting it
from the tensed state to relaxed state (T form to R form), and
increasing the sites available to the substrate. The alteration of
conformation of one subunit by substrate binding is transmitted
to other subunits by subunit interactions, and symmetry needn't
be conserved. Change induced by binding of substrate to one
subunit can increase or decrease substrate binding to other
subunits, i.e., the ligand-induced conformational change in one
subunit can affect the adjoining subunit. That means both
positive and negative homotropic interactions are possible in
this model. Heterotropic interactions could either be positive or
negative.

Figure 9: The sequential model for allosteric regulation

Reversible covalent modification

The activities of some regulatory enzymes are modulated by reversible covalent modification of the
enzyme molecule. These include the phosphorylation, adenylation, acetylation, uridylation, ADP-
ribosylation, and methylation of enzymes. The covalently attached groups are removed from the enzyme
by separate enzymes.
Phosphorylation

Phosphorylation (Figure 10) is the most common type of regulatory modification found in eukaryotes.
Some enzymes are phosphorylated on a single amino acid residue while others are phosphorylated at
multiple sites. To serve as an effective regulatory mechanism, phosphorylation must be reversible. In
general, phosphoryl groups are added and removed by different enzymes, and the processes can therefore
be separately regulated.
ATP ADP

P
Enzyme Enzyme

(Tyrosine, Serine, Threonine, Histidine)

Figure 10: Enzyme phosphorylation

The attachment of phosphoryl groups to specific

amino acid residues of a protein is catalyzed by protein
kinases; removal of phosphoryl groups is catalyzed by
protein phosphatases. The phosphoryl groups are
attached to serine, thereonine, or tyrosine residues,
thus introducing a bulky charged group into the
enzyme (figure-11).

Figure 11: Phosphorylation and dephosphorylation of amino acids

An important example of regulation by phosphorylation is observed in the enzyme glycogen

phosphorylase of muscle and liver, which catalyzes the following reaction:
Glycogen n + Pi → Glucose-1-phosphate + glycogen n-1

Glycogen phosphorylase is found in glycogen granules, which are optically dense bodies in the cytosol
containing all of the enzymes for synthesis and degradation of glycogen and some of the control elements.
This enzyme is typically an allosteric enzyme. This enzyme binds inorganic phosphate cooperatively. This
allows the enzyme’s activity to increase by great amounts over a narrow range of substrate concentrations.
Glycogen phosphorylase generates glucose-1-phosphate which is isomerized into glucose-6-phosphate and
enters the glycolytic pathway to produce ATP. This end product ATP is a feedback inhibitor of glycogen
phosphorylase. Glucose-6-phosphate is an allosteric inhibitor of the enzyme. ATP and glucose-6-phosphate
produce a negative effect on the cooperativity of substrate binding. AMP is also an allosteric effector of
glycogen phosphorylase. It competes for the same allosteric binding site as ATP but stimulates glycogen
phosphorylase by having a positive effect on the cooperativity of substrate binding. Increase in the cellular
concentration of AMP is an indicator that the energy status of the cell is low and more ATP via glycolysis
needs to be produced. The reciprocal changes of ATP and AMP concentrations combined with their
competition for the allosteric binding site with opposite effects provide a mechanism for rapid and reversible
control over glycogenolysis.

The allosteric controls allow the cell to adjust to normal metabolic demands. In crisis conditions in which
ATP is needed immediately, these allosteric controls are overridden by reversible covalent phosphorylation
of glycogen phosphorylase by the enzyme, phosphorylase kinase (Figure-12). The reversible covalent
modification of Ser-14 converts the enzyme from a less activated, allosterically regulated form b to a more
active, allosterically unresponsive form a. The phosphorylation of Ser-14 causes dramatic conformational
changes in phosphorylase. The phosphorylation of the serine residue essentially locks the enzyme into the
R-state. The phosphorylation is reversible (figure-12). The dephosphorylation is carried out by the enzyme
called phosphorylase phosphatase (PP-1).
 Figure 12: Regulation of the
enzyme glycogen phosphorylase

Phosphorylase a has two subunits, each with a specific serine (Ser) residue that is phosphorylated at its
hydroxyl group. These serine phosphate residues are required for maximum activity of the enzyme. The
phosphoryl groups can be hydrolytically removed by the enzyme phosphoprotein phosphatase and thus,
phosphorylase a is converted to phosphorylase b by the cleavage of two serine phosphate covalent bonds,
one on each subunit of glycogen phosphorylase.

Phosphorylase a + 2H2O → phosphorylase b + 2Pi

( less active)

As the process is reversible, phosphorylase b can be reactivated or covalently transformed back into
active phosphorylase a by the enzyme phosphorylase kinase, which catalyzes the transfer of phosphoryl
groups from ATP to the hydroxyl groups of the two specific Ser residues in phosphorylase b.

2ATP + Phosphorylase b → phosphorylase a + 2ADP

( more active)

The breakdown of glycogen in skeletal muscles and the liver is regulated by variations in the ratio of the
two forms of glycogen phosphorylase. The a and b forms differ in their secondary, tertiary and quaternary
structures; the active site undergoes changes in structure and consequently, changes in catalytic activity as
the two forms are interconverted.

The regulation of glycogen phosphorylase by phosphorylation illustrates the effects on both structure
and catalytic activity of adding a phosphoryl group. In the unphosphorylated state, each subunit of this
protein is folded so as to bring the twenty residues at its amino terminal end with a number of basic residues,
into a region containing several acidic amino acids which produces an electrostatic interaction that stabilizes
the conformation. Phosphorylation of Ser14 interferes with this interaction, forcing the amino-terminal
domain out of the acidic environment and into a conformation that allows interaction between the
phosphorylated serine and several arginine side chains. The enzyme is much more active in this
conformation.

As the number of known protein kinases has increased at an ever-accelerating pace, it has become more
challenging to determine which protein kinases interact with which substrates in the cell. The determination
of consensus phosphorylation site motifs by amino acid sequence alignment of known substrates has proven
useful in this pursuit. In the process of phosphorylating an amino acid, kinases recognize and bind to adjacent
amino acids towards both the N- and C-termini of the phosphorylation site, known as a consensus sequence.
The presence or absence of these consensus sequences determine whether a certain protein will be
phosphorylated by a given kinase or in other words, these consensus phosphorylation site motifs can be
helpful for predicting phosphorylation sites for specific protein kinases within a potential protein substrate.
Only three amino acids, i.e., serine, threonine, and tyrosine are the most commonly recognized as
phosphorylatable. The Ser, Thr, or Tyr residues that are phosphorylated in regulated proteins occur within
common structural motifs, i.e., called consensus sequences, that are recognised by specific protein kinases.
Some kinases are basophilic, preferring to phosphorylate a residue having basic neighbors; others have
different substrate preferences. Primary sequence is not the only important factor in determining whether
a given residue will be phosphorylated, the three-dimensional structure can determine whether a protein
kinase has access to a given residue and can recognise it as a substrate. In addition to the prediction of
phosphorylation sites, short synthetic oligopeptides based on consensus motifs are often excellent
substrates for protein kinase activity assays.

Regulation by phosphorylation often varies. Some proteins have consensus sequences recognised by
several different protein kinases, each of which can phosphorylate the protein and alters its enzymatic
activity. Some protein kinases contain phosphoamino acid residues in their recognition motifs, and have
been termed "hierarchical" protein kinases. They often require prior phosphorylation by another kinase at
a residue in the vicinity of their own phosphorylation site. For example, glycogen synthase, the enzyme
that catalyzes the condensation of glucose monomers to form glycogen, is inactivated by phosphorylation
of specific Ser residues and also modulated by at least four other protein kinases that phosphorylate four
other sites in the protein. The protein is not a substrate for glycogen synthase kinase 3, until one site has
been phosphorylated by casein kinase II. Glycogen synthase is directly regulated by glycogen synthase
kinase 3 (GSK-3), AMPK (AMP activated protein kinase) and protein kinase A (PKA). Each of these protein
kinases lead to phosphorylated and catalytically inactive glycogen synthase. Glycogen synthase is also
regulated by protein phosphatase 1 (PP1), which activates glycogen synthase via dephosphorylation. The
multiple regulatory phosphorylations provide the potential for extremely subtle modulation of enzymatic
activity. The phosphorylation sites of glycogen synthase are given in the table below (Table-1). The enzyme
has at least nine separate sites in five designated regions susceptible to phosphorylation by one of the
cellular protein kinases.

Table-1- The phosphorylation sites of the enzyme glycogen synthase

Kinase Phosphorylation sites Degree of synthase inactivation
Protein kinase A 1A, 1B, 2, 4 +
Protein kinase G 1A, 1B, 2 +
Protein kinase C 1A +
Ca2+/ Calmodulin kinase 1B, 2 +
Phosphorylase b kinase 2 +
Casein kinase I At least 9 ++++
Casein kinase II 5 0
Glycogen synthase kinase 3 3A, 3B, 3C +++
Glycogen synthase kinase 4 2 +

Adenylylation and Uridylylation

Adenylylation (figure-13) is the process in which adenosine-5 ‘ -monophosphate (AMP) is covalently

attached to a protein, nucleic acid, or small molecule via a phosphodiester or phosphoramidate linkage. In
the process of deadenylylation, AMP is removed from the adenylylated molecule. The adenylylation/
deadenylylation processes may provide regulatory control of enzyme activity, contribute to intermediate
steps in individual enzymatic reaction mechanisms, or occur as intermediate steps along the biosynthetic
pathway of cofactors. Adenylylation occurs in a wide range of organisms, including bacteria, yeast, and
mammals, although it is less common than many other post-translational modification reactions as a
source of enzyme regulation.

Uridylylation (fig-14) is the process in which, a uridylyl group is introduced into a protein, a ribonucleic acid,
or a sugar phosphate, generally through the action of a uridylyl transferase enzyme.
Uridylyltransferase is a single polypeptide chain. The Uridylyl transferase (UTase)/ uridylyl removing (UR)
enzyme catalyzes the uridylylation as well as the de-uridylylation of the regulatory protein PII, demonstrating
that a single bifunctional enzyme is involved in the covalent interconversion of PII. UTase and UR are
contained in single polypeptide. The uridylyltransferase/uridylyl-removing enzyme (UTase/UR) of Escherichia
coli plays an important role in the regulation of nitrogen assimilation by controlling the uridylylation state of
the PII signal transduction protein (PII) in response to intracellular signals. The reversible uridylylation of
PIIindirectly controls the activity of PII receptors that regulate transcription from nitrogen-regulated
promoters and the activity of glutamine synthetase.

In all organisms, glutamine synthetase (GS) plays a critical role in intermediary metabolism by catalyzing
the ATP-dependent condensation of ammonia with glutamate, to yield glutamine. Stadtman and co-workers
demonstrated that the differences in enzymatic activity of glutamine synthetase corresponded to the
presence or absence of covalently attached adenylyl groups. Regulation of glutamine biosynthesis is
characterized most thoroughly for E. coli and includes a complex bicyclic cascade that controls the
adenylylation of Tyr397 on the surface of GS. Bacterial GS are dodecameric oligomers with two face-to-face
hexameric rings. In the subset of bacterial strains that regulate GS activity via adenylylation, the
adenylylation state of GS may vary from 0 to 12 AMPs/GS dodecamer, and the enzymatic activity decreases
with increasing extent of adenylylation. Adenylylation and deadenylylation of the enzyme glutamine
synthase are both catalyzed by a single adenylyltransferase (ATase) whose activity is modulated by various
metabolites and by a regulatory protein, PII , which exists in two inter convertible forms PIIA and PIID. PIIA
and PIID stimulate adenylylation and deadenylylation activity of ATase respectively. PIIA is converted to PIID
by the presence of UTP, 2-oxoglutarate, ATP, and either Mg2+ or Mn2+. Mg2+ appeared to be the
physiologically relevant metal ion cofactor for both transferase and uridylyl-removing activities. High
intracellular levels of 2-oxoglutarate activate the uridylyltransferase portion of the bifunctional enzyme,
UTase/UR. The interconversion of PIIA to PIID involves covalent attachment of a uridine derivative to PII. The
covalently bound uridine derivative is UMP which is derived from UTP in a reaction catalyzed by the enzyme
uridylyltransferase (UTase).

Regulation of glutamine synthetase activity in E.

coli (figure-15) is thus facilitated by a highly
sophisticated cascade system of proteins,
consisting of an ATase, the regulatory protein
(PII), a signal transduction enzyme (Pn), and the
uridylyltransferase (UTase)/UR enzyme that
respond directly to nitrogen levels. When the
nitrogen levels are low, UTase/UR enzyme
uridylylates PII at Tyr51 to form PII-UMP. PII-UMP(
PIID) stimulates the deadenylylation activity of
ATase, to decrease the proportion of GS in the
adenylylated form and thus to increase the rate
at which nitrogen is "fixed" in the amino acid
glutamine,derived from glutamate by GS. When
Figure 15: The bicyclic cascade for regulation nitrogen levels are high, the UTase/UR-enzyme
of E.coli glutamine synthase (GS) cleaves the UMP from PII-UMP to generate
PII (PIIA).

PIIA stimulates the adenylylation activity of ATase, to increase the adenylylation state of GS and reduce
its efficiency in converting glutamate to glutamine. Thus, a high intracellular glutamine level activates the
uridylyl-removing portion of the bifunctional enzyme, UTase/UR. This in turn causes the de-uridylylation of
the regulatory protein PII. PII interacts with the enzyme adenylyltransferase which catalyzes the
adenylylation of glutamine synthetase and in effect inactivates it and therefore stopping the synthesis of
glutamine.
ADP-ribosylation

ADP-ribosylation (fig-16) is the addition of one or more ADP-ribose moieties to a protein. It is observed in
only a few proteins; the ADP-ribose is derived from nicotinamide adenine dinucleotide (NAD).

Figure 16: ADP-ribosylation

Arginine adenosine-5′-diphosphoribosylation (ADP-ribosylation) is an enzyme-catalyzed, potentially

reversible posttranslational modification, in which the ADP-ribose moiety is transferred from NAD+ to the
guanidino moiety of arginine, under release of nicotinamide (Figure 17). This reaction is catalyzed by a
subfamily of ADP-ribosyltransferases (ARTs), that bind NAD in an extended conformation, enabling the
nucleophilic attack of one of the two terminal nitrogen atoms of the guanidino group of arginine on the β-
N-glycosidic bond between nicotinamide and the C1′-atom of the ribose-group. Nicotinamide is released and
a new N-glycosidic bond between arginine and ADP-ribose is generated with an inversion of the
conformation at the C1′ atom of ADP-ribose from beta to alpha. Arginine ADP-ribosylation can be fully
reversed by specific enzymes ADP-ribosylhydrolases. Other acceptor amino acids, such as diphthamide (a
modified histidine), cysteine or asparagine, are targeted by other sub-families of ADP-ribosyltransferases via
a similar reaction mechanism.

Nitrogen fixation in some diazotrophic bacteria is regulated by mono-ADP-ribosylation of dinitrogenase

reductase (NifH) that occurs in response to addition of ammonium to the extracellular medium. This process
is mediated by dinitrogenase reductase ADP-ribosyltransferase (DraT) and reversed by dinitrogenase
reductase glycohydrolase (DraG). The ADP-ribosylation system has been identified in the purple, nonsulfur
photosynthetic bacteria R. rubrum and R. capsulatus, in the microaerophilic, associative
bacteria Azospirillum brasilense and Azospirillum lipoferum, and a purple sulfur bacterium Chromatium
vinosum. Although the means by which DraT and DraG are each regulated are not well understood, it is
known that the activity of each enzyme is regulated in vivo.

ADP-ribosylation is also responsible for the actions of some bacterial toxins, such as cholera toxin,
diphtheria toxin, and pertussis toxin. These toxin proteins are ADP-ribosyltransferases that modify target
proteins in human cells. For example, cholera toxin ADP-ribosylates a G proteins,that is a part of a signaling
pathway, which leads to several physiological responses and causing massive fluid secretion from the lining
of the small intestine, resulting in life-threatening diarrhea and sometimes causes death.
 Figure 17: Schematic diagram of the enzyme catalyzed,
reversible posttranslational modification of arginine by ADP-ribose

Methylation

Methylation is the addtion of a methyl group (Fig-18) to a protein. Enzyme regulation by methylation can be
observed in the methyl-accepting chemotaxis protein of bacteria. Methyl-accepting chemotaxis protein
(MCP) is a transmembrane sensor protein of bacteria. By the help of the MCPs, the bacteria can detect
concentrations of molecules in the extracellular matrix, as a result of which the bacteria may smooth swim
or tumble. The bacteria will have a forward swimming in response to a rising level of nutrients (attractants)
or decreasing level of toxins (repellents) and in response to a decreasing level of nutrients and increasing
level of toxins, the bacteria will tumble and reorient itself in a new direction. The transmembrane receptors,
MCPs may bind attractants or repellents directly or indirectly through interaction with proteins of
periplasmatic space. The signals from these receptors are transmitted across the plasma membrane into the
cytosol, where Che proteins are activated. The Che proteins alter the tumbling frequency, and alter the
receptors.

Figure 18: Methylation

MCPs undergo two covalent modifications: deamidation and reversible methylation at a number of
glutamate residues. Attractants and repellents change the methylation of the MCPs. Attractants increase
the level of methylation, while repellents decrease it. These methylation levels are the result of two
enzymatic processes: methyl groups are transferred from the methylating agent S-adenosylmethionine to
the MCPs by a methylation system, whereas methyl groups are removed by a demethylation system to
produce methanol. The methyl groups are added by the methyl-transferase cheR and are removed by the
methylesterase cheB. When the two systems are in balance, the level of methylation remains constant, but
the individual methyl groups turn over.
Proteolytic activation

The activation of an enzyme by peptide cleavage is known as proteolytic activation. In this enzyme regulation
process, the enzyme is shifted between the inactive and active state. Irreversible conversions can occur on
inactive enzymes to become active. This inactive precursor is known as a zymogen or a proenzyme, which is
cleaved to form the active enzyme. The cleavage is independent of ATP. This type of enzyme regulation is
different from allosteric regulation and reversible covalent modification of enzymes, as proteolytic
activation occurs just once in an enzyme's lifetime. So this type of enzyme regulation is also termed as
irreversible covalent modification.

Many proteolytic enzymes of the stomach and pancreas are regulated in this methodology. Blood
clotting is another example, which is carried out by a cascade of proteolytic activations. Certain protein
hormones are synthesizes as the inactive precursors such as proinsulin which then leads to the activated
form insulin by proteolytic cleavage. Collagen, a fibrous protein and the major component of skin and bone
is made from the zymogen procollagen. Programmed cell death, is mediated by proteolytic enzymes called
caspases, which are synthesized in precursor form as procaspases.

The digestive enzymes that hydrolyze proteins

are synthesized as zymogens in the stomach and
pancreas. Pepsinogen is the inactive protein,
which is cleaved in the stomach to produce pepsin,
a digestion enzyme. The purpose of this regulation
is to prevent the pepsin from digesting proteins in
the body before it is introduced into the digestive
tract. Chymotrypsinogen, the inactive form of the
enzyme chymotrypsin is synthesized in the
pancreas. Chymotrypsinogen consists of 245
amino acids. The cleavage of the bond between
the 15th amino acid (Arg) and the 16th amino acid
(Ile) by the enzyme trypsin (figure-19) activates the
enzyme chymotrypsinogen to chymotrypsin. The
two dipeptides are removed to produce α-
chymotrypsin. Three chains in α-chymotrypsin are
Figure 19: Proteolytic activation of chymotrypsinogen bonded by disulphide bonds.

Trypsinogen is the proenzyme which is activated to produce trypsin by the enzyme enteropeptidase,
which is a serine protease . To produce active trypsin, the cells that line the duodenum secrete an enzyme,
enteropeptidase, that hydrolyzes a unique lysine-isoleucine peptide bond in trypsinogen as the zymogen
enters the duodenum from the pancreas. The formation of trypsin by enteropeptidase is considered the
prime activaion step since trypsin participates in a variety of zymogen activation. Trypsin takes part in the
activation of proelastase to elastaase, procarboxypeptidase to carboxypeptidase, and prolipase to
lipase(figure-20).

Figure 20: Trypsin activates a variety of proenzymes

 Since proteolytic activation of enzymes is irreversible in nature, a different mechanism is needed to
stop proteolysis. These types of enzymes need very specific inhibitors which bind very tightly to the
enzyme active site. One example of which is pancreatic trypsin inhibitor, a 6-kd protein, inhibits trypsin by
binding very tightly to its active site. Another trypsin inhibitor such as α1-antitrypsin, also known as α1-
antiproteinase (antielastage), primarily inhibits neutrophil elastage. When the body contains excess
elastase, the enzyme destroys alveolar walls in the lungs by breaking down elastic fibers and other
connective tissue proteins in the lungs. This disease is called emphysema, containing symptoms such as
difficulty in breathing. Like pancreatic trypsin inhibitor, α1-antitrypsin blocks the action of target enzymes
by binding nearly irreversibly to their active sites.

Stimulation and inhibition by control proteins

Enzyme is regulated by binding of specific stimulatory or inhibitory protein. Protein phosphorylation, which
plays a key role in most cellular activities, is a reversible process mediated by protein kinases and
phosphoprotein phosphatases. Protein kinases catalyse the transfer of the gamma phosphate from
nucleotide triphosphates (often ATP) to one or more amino acid residues in a protein substrate side chain,
resulting in a conformational change affecting protein function. Phosphoprotein phosphatases catalyse the
reverse process. Cyclic AMP, an intercellular messenger, can activate protein kinase A (PKA) (figure-21). In
the absence of cAMP, Protein Kinase A (PKA) exists as an equimolar tetramer of regulatory (R) and catalytic
(C) subunits. Cyclic AMP activates Protein Kinase A by altering the quaternary structure.

Figure 21: Activation of protein kinase by cyclic AMP

The effects of cAMP in eukaryotic cells are due to activation of PKA by cAMP. The activation of that
multifunctional kinase is accomplished by cAMP binding to the regulatory subunit of the enzyme, and
release the catalytic units, releasing their inhibition of the catalytic subunits as well. The catalytic subunits
of PKA when they are released and active phosphorylate target substrate proteins on serine and threonine
residues, altering the activity of the modified protein and creating a cellular response to the extracellular
stimulus acting on the G-protein coupled receptors (GPCRs-one of the largest gene families of signaling
proteins).