Factors affecting enzyme activity:

Enzymes are generally globular proteins, acting alone or in larger complexes. Like all proteins,
enzymes are linear chains of amino acids that fold to produce a three-dimensional structure.
The sequence of the amino acids specifies the structure which in turn determines the catalytic
activity of the enzyme. Enzyme structures unfold (denature) when heated or exposed to
chemical denaturants and this disruption to the structure typically causes a loss of activity.
There many factors are influencing the activity of an enzyme are explained below.
The important factors affecting enzyme activity are:
1. Temperature
2. pH
3. Substrate concentration
4. Product concentration
5. Inhibitors
6. Activators

1. Temperature
Temperature influences the rate of reaction. At the ideal (optimum) temperature, the
chemical reactions will be very quick. These optimum temperatures are important for the
proper activity of the enzyme. which ranges between 37 to 40C° (Figure 1).

Figure 1: Affect of temperature on enzyme

• A higher temperature generally results in an increase in enzyme activity.
• As the temperature increases, molecular motion (entropy) increases resulting
in more molecular collisions.
• However, the temperature rises above a certain point (optimum temperature),
the heat will denature the enzyme, causing it to lose its three-dimensional
functional shape by denaturing its hydrogen bonds (irreversible damage).
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 • Cold temperature, on the other hand, slows down enzyme activity by
decreasing molecular motion.
• The enzyme activity stops completely at 0C°, but if the temperature rises again,
then the enzyme gets reactivated once more (reversible). Hence low
temperature will help in the storage of enzymes.
2. pH

Figure2: Affect of pH

• pH is a major factor affecting the activity of enzymes.

• The most favorable pH value - the point where the enzyme is most active - is
known as the optimum pH (Figure 2). Example: optimum pH of Pepsin is 1.5
- 1.6, Trypsin 7.8 - 8.7 and Amylase (pancreas) 6.7 - 7.0
• The ionization of amino acid molecules and atoms occurs when the pH is
altered, thereby affecting the structure and form of proteins and hence disrupting
their activities.
• The velocity of the enzyme is highest in the presence of its optimum pH, below
and above which the velocity is much lower.
• In case of the presence of extreme pH conditions, the enzyme is inactivated
altogether.
• The relationship between enzyme activity and pH is depicted by a bell-shaped
curve (Figure 2).

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3. Substrate concentration:
• As substrate concentration increases, there are more substrates available to bind with
the enzymes’ active sites in the same area of space.
• This means that the rate of reaction will increase as more products will be formed per
unit of time.
• However, once the substrate concentration has increased so much that all of the
enzyme’s active sites are occupied, increasing the concentration of substrate further
will have no effect.
• This is because there are no freely available enzyme active sites for them to bind to.
For this reason, the rate of reaction will remain the same, or plateau, as it cannot
increase any further (Figure 3).

Figure 3: Affect of substrate concentration on enzyme activity

4. Product concentration (Feedback inhibition)

• If the substrate is available in plenty, the formation of product increases product
concentration till they reach equilibrium.
• After the equilibrium, the end product accumulation retards the enzyme activity.
• When the product concentration rises above certain level, enough to be utilized by the
tissues, there is a control mechanism to avoid such wasteful production.
• In such a situation the product may interact with the enzyme to stop any further
synthesis of the product.
• Thus the product inhibits its own synthesis. This is called feedback inhibition or
allosteric modulation (Figure 4).

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 Figure 4: Affect of product on enzyme activity
5. Inhibitors
An enzyme inhibitor is a molecule that binds to an enzyme and either reduce the rate of enzyme
activity or blocks its activity. The enzyme inhibitors are among the most important
pharmaceutical agents known. For example, aspirin (acetylsalicylate) inhibits the enzyme that
catalyses the first step in the synthesis of prostaglandins, compounds involved in many
processes, including some that produce pain. The study of enzyme inhibitors also has provided
valuable information about enzyme mechanisms and has helped define some metabolic
pathways. There are two broad classes of enzyme inhibitors as mentioned below (Figure 5):
1. Reversible inhibition
2. Irreversible inhibition

Reversible inhibition:
In this type of inhibition, the inhibitor readily dissociates from the enzyme making it active
again. Reversible inhibitors attach to enzymes with non-covalent interactions such as hydrogen
bonds, hydrophobic interactions and ionic bonds. There are three types of inhibitors under
reversible inhibition.
1. Competitive inhibitor
2. Non-competitive
3. Uncompetitive inhibition

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 Figure 5: Types of inhibitors
1. Competitive inhibitor
• A competitive inhibitor competes with the substrate for the active site of an enzyme.
• While the inhibitor (I) occupies the active site, it prevents the substrate from binding to
the enzyme (Figure 6).
• Many competitive inhibitors are structurally similar to the substrate and combine with
the enzyme to form an EI complex, but without leading to catalysis.
• Even fleeting combinations of this type will reduce the efficiency of an enzyme.

Figure 6: Competitive inhibition

• For example, succinate dehydrogenase, a citric acid cycle enzyme that converts
succinate to fumarate. But succinate is competitively inhibited by malonate, because
malonate structurally resembles succinate but cannot be produce fumarate (Figure 7).

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 Figure 7: Example for competitive inhibition

2. Non-competitive inhibitor:
• In non-competitive inhibition the binding of the inhibitor other than active site of the
enzyme either free enzyme or enzyme substrate complex. This leads to reduction in
enzyme activity but does not affect the binding of substrate (Figure). As a result, the
extent of inhibition depends only on the concentration of the inhibitors (Figure 8).

Figure 8: Non-competitive inhibition

• Example for non-competitive inhibition: Cyanide action on cytochrome oxidase is an

example of non-competitive inhibition because cyanide acts as the inhibitor which
attaches to cytochrome oxidase at a place other than the active site and brings a
conformational change which in turn prevents the transport of electrons from
cytochrome c to oxygen.

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3. Un-competitive inhibition
• In uncompetitive inhibition, when an inhibitor molecule can bind to only the enzyme-
substrate complex but not to the free enzyme.
• Example for uncompetitive inhibition, inhibition of aryl sulphatase by hydrazine, and
another the inhibition of intestinal alkaline phosphatase by phenylalanine (Figure 9).

Figure 9: Un-competitive inhibition

Kinetics of enzyme inhibition:
Reversible inhibition can be described quantitatively in terms of the inhibitor's binding to the
enzyme and to the enzyme-substrate complex, and its effects on the kinetic constants of the
enzyme. In the classic Michaelis-Menten plot or Line weaver Burk plot, an enzyme (E) binds
to its substrate (S) to form the enzyme–substrate complex ES. Upon catalysis, this complex
breaks down to release product P and free enzyme. The inhibitor (I) can bind to either E or ES
with the dissociation constants Ki or Ki', respectively. The kinetics of the enzyme inhibitors
explained using Lineweaver Burk plot as follows:
• Competitive inhibitors can bind to E, but not to ES. Competitive inhibition increases
Km (i.e., the inhibitor interferes with substrate binding), but does not affect Vmax (the
inhibitor does not hamper catalysis in ES because it cannot bind to ES).
• Non-competitive inhibitors have identical affinities for E and ES (Ki = Ki'). Non-
competitive inhibition does not change Km (i.e., it does not affect substrate binding)
but decreases Vmax (i.e., inhibitor binding hampers catalysis)
• Uncompetitive inhibitors bind to ES. Uncompetitive inhibition decreases both Km and
Vmax. The inhibitor affects substrate binding by increasing the enzyme's affinity for

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 the substrate (decreasing Km) as well as hampering catalysis (decreases Vmax) (Figure
10).

Figure 10: Kinetics of enzyme inhibition explained using Lineweaver Burk plot

• Irreversible inhibition
Irreversible inhibitors covalently bind to an enzyme, and this type of inhibition can
therefore not be readily reversed. Irreversible inhibitors often contain reactive
functional groups such as nitrogen mustards, aldehydes, haloalkanes, alkenes, Michael
acceptors, phenyl sulfonates, or fluorophosphonates

Multi-substrate reactions: Ordered, Random, Ping-pong

In most enzymatic reactions, however, two (and sometimes more) different substrate molecules
bind to the enzyme and participate in the reaction. If the two substrates are involved in the
reaction, then it is called bi-substrate reactions. If more than two substrate involved in the
reaction, then it is called multi-substrate reaction. Nearly two-third of all enzymatic reactions
have two substrates and two products. These are generally reactions in which a group is
transferred from one substrate to the other, or one substrate is oxidized while the other is
reduced. For example, in the reaction catalyzed by hexokinase, ATP and glucose are the
substrate molecules, and ADP and glucose 6-phosphate are the products:
Nearly two-third of all enzymatic reactions have two substrates and two products.

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Enzymatic reactions with two substrates proceed by one of several different types of
pathways.
• Sequential Reactions
• Ping Pong Reactions

Sequential Reactions:
Reactions in which all substrates must combine with the enzyme before a reaction can occur
and products be released are known as sequential reactions. Enzymatic reactions with one,
two, three, or four substrates are referred to as uni-substrate reaction, bi-substrate reaction, ter-
substrate reaction, and quad-substrate reaction, respectively. Sequential displacement reactions
are further divided into two types:
• Ordered sequential reaction
• Random sequential reaction

Ordered sequential reaction:

➢ In the reaction, a compulsory order of substrate addition to the enzyme, which are said
to have an Ordered mechanism/ Ordered sequential reaction.
➢ In the Ordered mechanism, the binding of the first substrate is apparently required for
the enzyme to form the binding site for the second substrate.
➢ In a notation developed by W.W. Cleland, substrates are designated by the letters A and
B in the order that they add to the enzyme, products are designated by P and Q in the
order that they leave the enzyme, the enzyme is represented by a horizontal line, and
successive additions of substrates and releases of products are denoted by vertical
arrows (Figure 11).

Figure 11: An Ordered bi-substrate reaction

where A and B are said to be the leading and following substrates, respectively

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Significance: Many NAD+- and NADP+-requiring dehydrogenases follow an Ordered bi-
substrate mechanism.

Random sequential reaction:

➢ In the reaction, no preference for the order of substrate addition to the enzyme, which
are called Random mechanism/Random sequential reaction.
➢ In the Random mechanism, both binding sites are present on the free enzyme.
➢ According to the W.W. Cleland notation, substrates are designated by the letters A and
B in the order that they add to the enzyme, products are designated by P and Q in the
order that they leave the enzyme, the enzyme is represented by a horizontal line, and
successive additions of substrates and releases of products are denoted by vertical
arrows (Figure 12).

Figure 12: An random bi-substrate reaction

Significance: Some dehydrogenases and kinases operate through Random bisubstrate
mechanisms (kinases are enzymes that transfer phosphoryl groups from ATP to other
compounds or vice versa).

Ping Pong Reactions

In the reaction, one or more products are released before all substrates have been added are
known as Ping Pong reactions.

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 Figure 13: Ping-Pong bi-substrate reaction
*F=stable enzyme
• Here, the first substrate A is displaced from the substrate by the enzyme E to yield the
first product P and a stable enzyme form F (Ping).
• In the second stage of the reaction, stable enzyme (F) binding with the second substrate
B to yield the second product Q, thereby regenerating the original form of the enzyme,
E (Pong). Such reactions are therefore also known as double-displacement reactions
(Figure 13).
• Example: trypsin (in which F is the acyl–enzyme intermediate), transaminases, and
some flvoenzymes, react with Ping Pong mechanisms

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 Enzyme regulation

Allosteric enzyme
Allosteric enzymes are enzymes that have an additional binding site for effector (modulator)
molecules other than the active site. The term “allosteric” derives from the Greek allos,
“other,” and stereos, “solid” or “shape.” The binding brings about conformational changes,
thereby changing its catalytic properties. The effector (modulator) molecule can be an
inhibitor or activator (Figure 14). All the biological systems are well regulated. There are
various regulatory measures in living cells, that control all the processes and respond to the
various inside and outside environmental changes. Whether it is gene expression, cell division,
hormone secretion, metabolism or enzyme activity, everything is regulated to ensure proper
development and survival.

Figure 14: structure Allosteric enzyme and their effector molecules

Example for allosteric enzyme: Aspartate transcarbamoylase (ATCase) from E. coli.

Aspartate transcarbamoylase catalyzes the formation of N-carbamoyl aspartate from
carbamoyl phosphate and aspartate (substrate). The allosteric behaviour of E. coli ATCase has
been investigated by John Gerhart and Howard Schachman, who demonstrated that both of
its substrates bind cooperatively to the enzyme. Moreover, ATCase is allosterically inhibited

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by cytidine triphosphate (CTP), a pyrimidine nucleotide, and is allosterically activated by
adenosine triphosphate (ATP), a purine nucleotide

Figure 15: Example for allosteric enzyme modulation: The Feedback Inhibition of ATCase
Regulates Pyrimidine Synthesis

Allosteric Enzyme Properties

1. They have multiple (one or more) allosteric sites.
2. Allosteric sites are binding sites that are unique from an enzyme active site for
binding sites for substrates.
3. The molecules that bind to the allosteric site are modulators or effectors. The activators
in allosteric enzymes which increase activity enzymes, whereas inhibitors decrease the
activity of enzymes.
4. Effector may be positive or negative, this effector regulates the enzyme activity. The
enzyme activity is increased when a positive allosteric effector binds at the allosteric
site known as activator site. On the other hand, negative allosteric effector binds at the
allosteric site called inhibitor site and inhibit the enzyme activity.
5. Binding to allosteric sites alter the activity of the enzyme, this is called cooperative
binding. Allosteric enzymes display sigmoidal plot of V₀ vs [S].
6. The enzyme is heterotrophic when the modulator has a structure different from the
substrate.
7. When the regulatory enzyme of a substrate and the modulator are identical, it is known
as homo-tropic.

Mechanisms of allosteric enzyme regulations

The allosteric enzymes have conformational changes induced by one or more modulators
interconvert more-active and less-active forms of the enzyme. The modulators for allosteric
enzymes may be inhibitory or stimulatory.
The allosteric enzymes can be regulated on the basis of two types i.e one for substrate and other
for effector molecules.

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Two types of allosteric regulation are:
1. Homotropic Regulation: In this type of regulation substrate molecules act as an
effector also. They are mainly enzyme activation and known as cooperativity. Example
of homotropic regulation is binding of oxygen to haemoglobin.

2. Heterotropic Regulation: This is a kind of regulation where substrate and effector are
different. Example of heterotropic regulation is binding of carbon dioxide (CO2) to
haemoglobin.

Figure16: Allosteric regulations

On the basis of the above action performed by the regulator, there are two types of
regulation one is activator and other is inhibitors.
Allosteric Inhibition: Under this process inhibitors bind with protein due to which all
active sites of protein undergo conformational changes due to which activity of enzyme
decreases.
Allosteric Activation: Under this process activator binds with protein which increases
the function of active sites leads to increase in enzymatic activity.

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Models Based of Allosteric Enzymes Regulation

There are so many modals which are proposed on mechanism of allosteric enzymes, out of
which two models that account for cooperative ligand binding have received the most attention.
One of them, the symmetry model of allosterism, formulated in 1965 by Jacques Monod,
Jeffries Wyman, and Jean-Pierre Changeux,(MWC) is defined by the following rules:

1. An allosteric protein is an oligomer of symmetrically related subunits (although the

α and β subunits of hemoglobin are only pseudosymmetrically related).
2. Each oligomer can exist in two conformational states, designated R (relaxed) and T
(inactive); these states are in equilibrium.
3. The ligand can bind to a subunit in either conformation. Only the conformational
change alters the affinity for the ligand.
4. The molecular symmetry of the protein is conserved during the conformational change.
The subunits must therefore change conformation in a concerted manner; in other
words, there are no oligomers that simultaneously contain R- and T-state subunits.

Figure 17 : The symmetry model of allosterism. Squares and circles represent T- and R-state
subunits, respectively, of a tetrameric protein. The T and R states are in equilibrium regardless
of the number of ligands (represented by S) that have bound to the protein. All the subunits
must be in either the T or the R form; the model does not allow combinations of T and R-state
subunits in the same protein.

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• All subunits are either in the active form (R) or all are in inactive form(T).
• Every substrate molecule that binds with enzyme increases the probability of transition from
the inactive to the active site.
• The effect of allosteric activators and inhibitors can be explained quite easily by this model.
• An allosteric inhibitor binds preferably to the T form whereas an allosteric activator binds
to the R form.
• An allosteric inhibitor shifts The R → T conformational equilibrium towards T. Whereas an
allosteric activator shifts it toward R.
• The result is that an allosteric activator increases the binding to substrate of the enzyme,
whereas an allosteric inhibitor decreases substrate binding.

Limitation of MWC Model

1. One major objection to the symmetry model is that it is difficult to believe that
oligomeric symmetry is perfectly preserved in all proteins, that is, that the T → R
shift occurs simultaneously in all subunits regardless of the number of ligands
bound.
2. In addition, the symmetry model can account only for positive cooperativity,
although some proteins exhibit negative cooperativity

An alternative to the symmetry model is the sequential model of allosterism, proposed by

Daniel Koshland, Némethy and Filmer in 1966

Figure 18: The sequential model of allosterism.

• In this model, the binding of substrate induces a change in the conformation of the
enzyme from T (tensed) to R (relaxed) within the oligomerse

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 • The substrate binds according to the induced fit theory. A conformational change in
one unit stimulates similar changes in other subunits. This explains the cooperative
binding.
• The same way inhibitors and activators bind, the T form is favoured, when the inhibitor
binds and R form is favoured, when the activator binds. The binding at one subunit
affects the conformation of other subunits.
• The sequential model explains the negative cooperativity in enzymes, e.g. tyrosyl tRNA
synthetase, where the binding of substrate inhibits the binding of another substrate.
• In addition, this model can account both for positive cooperativity and negative
cooperativity

Hills equation
• Enzyme Cooperativity is a phenomenon in which the shape of one subunit of an
enzyme consisting of several subunits is altered by the binding of the ligand.
• Cooperative binding of oxygen by hemoglobin was first analyzed by Archibald Hill
in 1910.
• This ligand can be either the substrate itself or some other molecule which alters the
substrate binding on the enzyme.
• If the ligand induced change in shape facilitates the binding of substrate to the same or
other subunit, the effect is called positive cooperativity.
• In negative cooperativity, the binding of a molecule to the first subunit inhibits the
binding of substrate to the same or other subunit.
• Therefore, in multi subunit enzymes, binding of substrate is cooperative and it shows
sigmoidal curve unlike the hyperbolic curve in Michaelis-Menten model
• These enzymes consist of multiple subunits and multiple active sites with binding
ability of substrates at different binding sites.
• In allosteric enzymes, the binding of substrate to one active site can affect the
properties of other active sites in the same enzyme molecule. Due to this reason, the
binding of substrate becomes cooperative; that is, the binding of substrate to one active
site of the enzyme facilitates substrate binding to the other active sites.
• For enzymes that display positive cooperativity in binding ligand usually display
sigmoidal (because it resembles "S") curve, when the curve is plotted between the

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 reaction velocity V0 and ligand concentration [L] (Figure 19); as compared to the
hyperbolic plots predicted by the Michaelis-Menten model.
• Hills equation represented:

Where L: concentration of ligand, Kd: Hill’s constant, n: Hill coefficient

n = 1, no cooperative
n>1 positive cooperativity
n<1 negative cooperativity

Figure 19: Hill’s plot: positive cooperativity in binding substrate

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