Kinetics of enzyme action,
allosteric effects.
Dr. J.O.Akande
Introduction
• Life is possible due to the coordination of numerous metabolic reactions
inside the cells. Proteins can be hydrolyzed with hydrochloric acid by
boiling for a very long time; but inside the body, with the help of
enzymes, proteolysis takes place within a short time at body
temperature.
• Enzyme catalysis is very rapid; usually molecule of an enzyme can act
upon about 1000 molecules of the substrate per minute.
• Lack of enzymes will lead to block in metabolic pathways causing inborn
errors of metabolism.
• The substance upon which an enzyme acts, is called the substrate.
• The enzyme will convert the substrate into the product or products.
Characteristics of Enzymes
I.
II.
III.
IV.
V.
Almost all enzymes are proteins. Enzymes follow the physical and
chemical reactions of proteins.
They are heat labile.
They are water-soluble.
They can be precipitated by protein precipitating reagents (ammonium
sulfate or trichloroacetic acid).
They contain 16% weight as nitrogen.
CLASSIFICATION OF ENZYMES
• Class 1: Oxidoreductases: Transfer of hydrogen or addition of oxygen; e.g.
Lactate dehydrogenase (NAD); Glucose-6-phosphate dehydrogenase (NADP);
Succinate dehydrogenase (FAD); dioxygenases.
• Class 2: Transferases: Transfer of groups other than hydrogen. Example,
Aminotransferase. (Subclass: Kinase, transfer of phosphoryl group from ATP;
e.g. Hexokinase).
• Class 3: Hydrolases: Cleave bond and add water; e.g. Acetylcholine esterase;
Trypsin.
• Class 4: Lyases: Cleave without adding water, e.g. Aldolase; HMG CoA lyase; ATP
Citrate lyase. (Subclass: Hydratase; add water to a double bond).
• Class 5: Isomerases: Intramolecular transfers. They include racemases and
epimerases. Example, Triose phosphate isomerase.
• Class 6: Ligases: ATP-dependent condensation of two molecules, e.g. Acetyl CoA
carboxylase; Glutamine synthetase; PRPP synthetase.
CO-ENZYMES
I.
Enzymes may be simple proteins, or complex enzymes, containing a nonprotein part, called the prosthetic group.
• The prosthetic group is called the co-enzyme. It is heat stable.
II. The protein part of the enzyme is then named the apoenzyme.
• It is heat labile.
III. These two portions combined together are called the holo-enzyme.
IV. Co-enzymes may be divided into two groups;
a. Those taking part in reactions catalyzed by oxidoreductases by donating or
accepting hydrogen atoms or electrons.
b. Those co-enzymes taking part in reactions transferring groups other than hydrogen.
Cont’d
1. The protein part of the enzyme gives the necessary three dimensional
infrastructure for chemical reaction; but the group is transferred from or accepted
by the co-enzyme
2. The co-enzyme is essential for the biological activity of the enzyme
3. Co-enzyme is a low molecular weight organic substance. It is heat stable
4. Generally, the co-enzymes combine loosely with the enzyme molecules. The
enzyme and co-enzyme can be separated easily by dialysis
5. Inside the body, when the reaction is completed, the coenzyme is released from
the apo-enzyme, and can bind to another enzyme molecule., the reduced coenzyme, generated in the first reaction can take part in the second reaction. The
coupling of these two reactions becomes essential in anaerobic glycolysis for
regeneration of NAD+
6. One molecule of the co-enzyme is able to convert a large number of substrate
molecules with the help of enzyme
7. Most of the co-enzymes are derivatives of vitamin B complex substances.
Examples of co-enzymes
Co-enzyme
Group transferred
Thiamine pyrophosphate (TPP)
Hydroxyethyl
Pyridoxal phosphate (PLP)
Amino group
Biotin
Carbon dioxide
Co-enzyme-A (Co-A)
Acyl groups
Tetrahydrofolate (FH4)
One carbon groups
Adenosine triphosphate (ATP)
Phosphate
Metallo-enzymes
I.
These are enzymes, which require certain metal ions for their
activity. Some examples are given in
II. In certain cases, e.g. copper in Tyrosinase, the metal is tightly
bound with the enzyme.
III. In other cases, even without the metal ion, enzyme may be active;
but when the metal ion is added, the activity is enhanced. They are
called ion-activated enzymes, e.g. calcium ions will activate
pancreatic lipase.
• NB: Co-factors
• The term co-factor is used as a collective term to include coenzymes
and metal ions.
• Co-enzyme is an organic co-factor.
Cont’d
Metal Enzymes containing metals
Metal Enzymes containing metals
Zinc
Carbonic anhydrase, carboxy peptidase, alcohol
dehydrogenase
Magnesium
Hexokinase, phosphofructokinase, enolase,
glucose-6-phosphatase
Manganese
Phosphoglucomutase, hexokinase, enolase,
glycosyl transferases
Copper
Tyrosinase, cytochrome oxidase, lysyl oxidase,
superoxide dismutase
Iron
Cytochrome oxidase, catalase, peroxidase,
xanthine oxidase
Calcium
Lecithinase, lipase
Molybdenum
Xanthine oxidase
Cont’d
• Nonprotein components required for the enzymatic activity: cofactor
– Apoenzyme + cofactor = holoenzyme
– Two types of cofactors:
• Metal ions: Mg2+, Zn2+, Cu2+, Mn2+, ...
• Coenzymes: small organic molecules
synthesised from vitamins.
Prosthetic groups: tightly bound coenzymes
• Cofactors deficiency promotes some health problems.
MODE OF ACTION OF ENZYMES
• Lowering of Activation Energy
I. Enzymes lower the energy of activation.
II. Activation energy is defined as the energy required to convert all molecules of a
reacting substance from the ground state to the transition state.
III. Substrates are remaining in an energy trough, and are to be placed at a higher
energy level, whereupon spontaneous degradation can occur. Suppose, we want
to make a fire; even if we keep a flame, the wood will not burn initially; we have
to add kerosene or paper for initial burning. Similarly, the activation energy is to
be initially supplied.
IV. During enzyme substrate binding, weak interactions between enzyme and
substrate are optimized. This weak binding interaction between enzyme and
substrate provides the major driving force for the enzymatic catalysis.
V. Enzymes reduce the magnitude of this activation energy. This can be compared to
making a tunnel in a mountain, so that the barrier could be lowered.
• For example, activation energy for acid hydrolysis of sucrose is 26,000 cal/mol, while
the activation energy is only 9,000 cal/mol when hydrolyzed by sucrase.
Entropy Effect
• Enzymes enhance reaction rates by decreasing entropy. When correctly
positioned and bound on the enzyme surface, the substrates are
strained to the transition state. This is referred to as the Proximity effect.
Chemical reactions need physical apposition of two reactants.
• The occurrence of collision between two substrate molecules is
determined by statistical probability. Since substrates usually are present
in low concentrations, the collision probability is less and hence the
reaction velocity is low.
• But a complex formation between the enzyme and the two substrate
molecules can improve the collision probabilities many fold, causing the
rapid rate of reaction.
Cont’d
MICHAELIS-MENTEN THEORY
I.
In 1913, Michaelis and Menten put forward the Enzyme-Substrate complex
theory. Accordingly, the enzyme (E) combines with the substrate (S), to
form an enzyme-substrate (ES) complex, which immediately breaks down to
the enzyme and the product (P)
E + S →E–S Complex → E + P
II. Alkaline phosphatase hydrolyzes a number of phosphate esters including
glucose-6-phosphate. The active Centre of this enzyme contains a Serine
residue, and the reaction is taking place in the following two
• steps:
a. E-Serine-OH+Glucose-6-P→E-Serine-O-P+Glucose
b. E-Serine-O-P → E-Serine-OH+Pi
• Thus, the overall reaction is Glucose-6-P → Glucose + Pi
• In this reaction mixture, the enzyme-substrate complex, E-Serine-O-P, has been isolated.
For Michaelis -Menton kinetics k2= kcat
When [S] << KM very little ES is formed and [E] = [E]T
and
k cat
k2
E T S  E S
vo 
KM
KM
Kcat/KM is a measure of catalytic efficiency
Cont’d
FISCHER'S TEMPLATE THEORY
I.
It states that the threedimensional structure of the
active site of the enzyme is
complementary to the substrate.
II. Thus enzyme and substrate fit
each other. The substrate fits on
the enzyme, similar to a lock and
key. The lock can be opened by
its own key only.
III. However, Fischer envisaged a
rigid structure for enzymes,
which could not explain the
flexibility shown by enzymes.
KOSHLAND'S INDUCED FIT THEORY
I.
Conformational changes are occurring at the active site of enzymes
concomitant with the combination of enzymes with the substrate. At
first, the substrate binds to a specific part of the enzyme.
II. This leads to more secondary binding and conformational changes. The
substrate induces conformational changes in the enzyme, such that
precise orientation of catalytic groups is affected. A simplified
explanation is that a glove is put on a hand. At first, the glove is in a
partially folded position, but hand can enter into it. When the hand is
introduced, the glove is further opened. Similarly, conformational
changes occur in the enzyme when the substrate is fixed.
III. When substrate analog is fixed to the enzyme, some structural
alteration may occur; but reaction does not take place due to lack of
proper alignment. Allosteric inhibition can also be explained by the
hypothesis of Koshland.
Cont’d
1 Enzyme has shallow grooves;
substrate alignment is not correct.
2. Fixing of substrate induces
structural changes in enzyme.
3. Now substrate correctly fits into
the active site of enzyme.
4. Substrate is cleaved into two
products.
Enzyme-substrate interactions
Fischer:
Lock & key
Koshland:
Induced fit
3a. Physical bond strain
Draw an quarter - an anvil
ACTIVE SITE OR ACTIVE CENTER OF ENZYME
1.
2.
3.
4.
5.
6.
7.
The region of the enzyme where substrate binding and catalysis occurs is referred to
as active site or active center.
Although all parts are required for maintaining the exact three-dimensional structure
of the enzyme, the reaction is taking place at the active site. The active site occupies
only a small portion of the whole enzyme.
Generally, active site is situated in a crevice or cleft of the enzyme molecule. To the
active site, the specific substrate is bound. The binding of substrate to active site
depends on the alignment of specific groups or atoms at active site.
During the binding, these groups may realign themselves to provide the unique
conformational orientation so as to promote exact fitting of substrate to the active
site.
The substrate binds to the enzyme at the active site by noncovalent bonds. These
forces are hydrophobic in nature.
The amino acids or groups that directly participate in making or breaking the bonds
(present at the active site) are called catalytic residues or catalytic groups
The active site contains substrate binding site and catalytic site; sometimes these two
may be separate.
• The catalytic cycle of an enzyme
1 Substrates enter active site; enzyme
changes shape so its active site
embraces the substrates (induced fit).
Substrates
Enzyme-substrate
complex
6 Active site
Is available for
two new substrate
Mole.
Enzyme
5 Products are
Released.
Figure 8.17
Products
2 Substrates held in
active site by weak
interactions, such as
hydrogen bonds and
ionic bonds.
3 Active site (and R groups of
its amino acids) can lower EA
and speed up a reaction by
• acting as a template for
substrate orientation,
• stressing the substrates
and stabilizing the
transition state,
• providing a favorable
microenvironment,
• participating directly in the
catalytic reaction.
4 Substrates are
Converted into
Products.
Cont’d
Name of enzyme
Important amino acid at
the catalytic site
Chymotrypsin
His (57), Asp (102), Ser (195)
Trypsin
Serine, Histidine
Thrombin
Serine, Histidine
Phosphoglucomutase
Serine
Alkaline phosphatase
Serine
Acetylcholinesterase
Serine
Carbonic anhydrase
Cysteine
Hexokinase
Histidine
Carboxypeptidase
Histidine, Arginine, Tyrosine
Aldolase
Lysine
THERMODYNAMIC CONSIDERATIONS
• From the standpoint of energy, the enzymatic
reactions are
divided into 3 types:
• Exergonic or Exothermic Reaction
• Here energy is released from the reaction,
and therefore reaction essentially goes to
completion, e.g. urease enzyme:
• Urea → ammonia + CO2 + energy
• At equilibrium of this reaction, the substrate
will be only 0.5% and product will be 99.5%.
Such reactions are generally irreversible.
• Isothermic Reaction
• When energy exchange is negligible, the
reaction is easily reversible, e.g.
• Glycogen + Pi → Glucose-1-phosphate. At
equilibrium of this reaction, 77% glycogen will
be unutilized and 23% glucose-1-phosphate
will be formed.
• Endergonic or Endothermic Reaction
• Energy is consumed and external energy is to
be supplied for these reactions. In the body,
this is usually accomplished by coupling the
endergonic reaction with an exergonic
reaction, e.g. Hexokinase catalyzes the
following reaction:
• Glucose + ATP → Glucose-6-Phosphate + ADP
ENZYME KINETICS
• Velocity or rate of enzyme reaction is assessed by the rate of change of substrate to product per unit time.
• In practice, initial velocity is determined. If much time is allowed to lapse, the velocity may tend to fall due to decrease
in substrate concentration below a critical level.
• The velocity is proportional to the concentration of reacting molecules.
• A+B→C+D
• If concentration of A or B is doubled, the rate of reaction is also doubled. If concentrations of A and B are doubled
together, the velocity becomes 4-fold.
1. The equilibrium constant of the reaction is the ratio of reaction rate constants of forward and backward reactions.
2. At equilibrium, forward and backward reactions are equal. Equilibrium is a dynamic state. Even though no net
change in concentrations of substrate and product occurs, molecules are always interconverted.
3. Numerical value of the constant can be calculated by finding the concentrations of substrates and products.
4. If Keq is more than 1, the forward reaction is favored. In such instances, the reaction is spontaneous and
exothermic.
5. Concentration of enzyme does not affect the Keq. Concentration of enzyme certainly increases the rate of reaction;
but not the Keq or the ultimate state. In other words, enzyme makes it quicker to reach the equilibrium.
• Catalysts increase the rate of reaction, but do not alter the equilibrium.
FACTORS INFLUENCING ENZYME ACTIVITY
1. Enzyme concentration
2. Substrate concentration
3. Product concentration
4. Temperature
5. Hydrogen ion concentration (pH)
6. Presence of activators
7. Presence of inhibitors
8. Presence of repressor or derepressor
9. Covalent modification.
Enzyme Concentration
I.
Rate of a reaction or velocity (V) is directly proportional to the enzyme
concentration, when sufficient substrate is present. The velocity of the
reaction is increased proportionately with the concentration of
enzyme, provided substrate concentration is unlimited.
II. Hence, this property is made use of determining the level of particular
enzyme in plasma, serum or tissues.
III. Known volume of serum is incubated with substrate
IV. for a fixed time, then reaction is stopped and product is quantitated
(end point method). Since the product formed will be proportional to
the enzyme concentration, the latter could be assayed.
Cont’d
Effect of enzyme concentration
Effect of substrate concentration
(substrate saturation curve
Effect of Substrate Concentration on Reaction Rate
• The effect on V0 of varying [S] when the enzyme concentration is held constant
• This is the appearance of a V0 vs [S] kinetic plot for a typical enzyme.
• At relatively low concentrations of substrate, V0 increases almost linearly with an increase in [S].
• At higher substrate concentrations, V0 increases by smaller and smaller amounts in response to
increases in [S].
• Finally, a point is reached beyond which increases in V0 are vanishingly small as [S] increases. This
plateau-like V0 region is close to the maximum velocity, Vmax.
Effect of substrate concentration on enzyme
activity
Enzyme pH-activity Profiles
Enzymes have an optimum pH at which their activity is maximal. At higher or
lower pH values, their activity declines .
This is because ionizable amino acid side-chains that are important for catalysis
of the reaction, or maintain the structure of the enzyme, must maintain a certain
state of ionization to function properly.
The pH range over which an enzyme undergoes changes in activity can provide a
clue as to the type of amino acid residue involved in catalysis. A change in
activity near pH 7.0, for example, often reflects titration of a His residue.
However, the effects of pH on activity must be interpreted cautiously, as in the
closely packed environment of a protein, the pKa of an amino acid side-chain can
vary significantly from the pKa of the free amino acid in solution.
The pH optimum for the activity of an enzyme generally is close to that of the pH
of the environment in which the enzyme normally functions. For example, the
pH optimum of pepsin, a gastric digestive enzyme, is about 1.6. The pH optimum
of the cytoplasmic enzyme, glucose 6-phosphatase, of hepatocytes is about 7.8.
Enzyme Inhibition
• Enzyme inhibitors are molecules that interfere with catalysis, slowing or halting enzymatic
reactions.
• Enzyme inhibitors are among the most important pharmaceutical agents known.
• For example, aspirin (acetylsalicylate) inhibits the enzyme that catalyzes the first step in the
synthesis of prostaglandins, compounds involved in many processes, including some that cause
pain.
• The study of enzyme inhibitors also has provided valuable information about enzyme mechanisms
and has helped define metabolic pathways.
• There are two broad classes of enzyme inhibitors: reversible and irreversible inhibitors.
Competitive Inhibition
• One example of reversible enzyme inhibition will be covered: competitive
inhibition.
• A competitive inhibitor (I) competes with the substrate for binding to the
active site of an enzyme.
• While the inhibitor occupies the active site, it prevents the binding of the
substrate to the enzyme and blocks the reaction. Many competitive inhibitors
are structurally similar to the substrate and combine with the enzyme to form
an EI complex, but without leading to catalysis. Competitive inhibition can be
analyzed quantitatively by steady-state kinetics. In the presence of a
competitive inhibitor, the MM equation becomes
V0 = Vmax[S]/(Km + [S])
Where
 = 1 + [I]/KI and KI = [E][I]/[EI].
The experimentally determined variable Km,
the Km observed in the presence of the
competitive inhibitor, is often called the
“apparent” Km.
Cont’d
Because a competitive inhibitor binds reversibly to an
enzyme, the competition can be biased to favor the
substrate simply by adding more substrate to the reaction.
When [S] far exceeds [I], the probability that an inhibitor
will bind to the enzyme is minimized and the reaction
exhibits a normal Vmax.
However, the [S] at which V0 = 1/2 Vmax, the apparent Km,
increases in the presence of inhibitor by the factor .
This affect on apparent Km, combined with the absence of
an effect on Vmax, is diagnostic of competitive inhibition
and is readily revealed in a double-reciprocal kinetic plot.
The equilibrium constant for inhibitor binding, KI, can also
be obtained from these plots. Many drugs act by
competitively inhibiting enzymes (e.g., ibuprofen and the
cyclooxygenase enzymes, COX 1 & 2).
Irreversible Inhibition
• Irreversible inhibitors bind covalently to or
destroy a functional group on an enzyme
that is essential for the enzyme’s activity.
They also can inhibit an enzyme by
forming a particularly stable noncovalent
association with the enzyme.
• An example of a irreversible covalent
inhibitor of the protease, chymotrypsin. As
we will discuss in the next lecture slide
file, chymotrypsin contains a reactive
serine residue in its active site that is
intimately involved in catalysis of peptide
bond cleavage.
• This serine will react with the inhibitor
diisopropylfluorophosphate (DIFP) which
modifies the serine residue irreversibly,
and thereby inhibits the proteolytic
activity of the enzyme.
• In contrast to ibuprofen, aspirin is a
covalent irreversible inhibitor of COX
enzymes.
Mechanism-based Inactivators
• A special class of irreversible inhibitors are the mechanism-based (suicide) inactivators.
• These compounds are relatively unreactive until they bind to the active site of a specific
enzyme.
• A suicide inactivator undergoes the first few chemical steps of the normal enzymatic
reaction, but instead of being transformed into the normal product, the inactivator is
converted into a very reactive compound that combines irreversibly with the enzyme.
• These inhibitors earn their name because they hijack the normal enzyme reaction
mechanism to inactivate the enzyme. Because drugs that serve as mechanism-based
inactivators are highly specific for their target enzymes, they often have the advantage
of few side effects.
• An example of a mechanism-based inhibitor that is used in the treatment of the
disease, trypanosomiasis, is presented in Box 6-3 (not covered).
Transition-state Analogs
An irreversible enzyme inhibitor need not bind
covalently to an enzyme if noncovalent binding is
so tight that the inhibitor dissociates only rarely.
Such inhibitors commonly resemble the predicted
transition state structure of the reaction and are
called transition-state analogs.
These compounds bind more tightly to an enzyme
than the substrate because they fit into the active
site better.
For example, transition state analogs designed to
inhibit the glycolytic enzyme aldolase bind to that
enzyme more than four order of magnitude more
tightly than its actual substrates.
Observations that such molecules are effectively
irreversible inhibitors of enzymes, support the
concept that enzyme active sites are most
complementary to that of the transition state of
the reaction.
Lastly, anti-HIV drugs that inhibit the required
protease function of the virus are actually
transition-state analogs.
Competitive
Inhibitors:
bind to active site
“unproductively”
and block
true substrates’
access
HO
S2
S1
OH
OH
-
I
HO
OH
OH
HO
HO
S & I bind to same site
+
Competitive inhibition
Allosteric Inhibitors
“other” “site”
Distorts the conformation
of the enzyme
Negative
allosteric
regulator
Allosteric inhibition
Positive allosteric regulators
Helps enzyme work better
promotes/stabilizes an “active” conformation
Allosteric activation
Allosteric regulators change the shape
conformation of the enzyme
Allosteric enyzme
with four subunits
Regulatory
site (one
of four)
Active site
(one of four)
Activator
Active form
Stabilized active form
Oscillation
Allosteric activater
stabilizes active form
NonInactive form Inhibitor
functional
active
site
Figure 8.20
Allosteric activater
stabilizes active from
Stabilized inactive
form
(a) Allosteric activators and inhibitors. In the cell, activators and inhibitors
dissociate when at low concentrations. The enzyme can then oscillate again.
A frequent regulatory modification
Phosphorylation of enzymes
Phosphorylase kinase
inactive
+ P
active
Summary
•
•
•
•
•
•
•
enzymes are catalysts
Lower activation energy EA
Mechanism of action …
Enzyme kinetics- Vmax, Km
Regulation of enzyme activity
- competitive, allosteric
phosphorylation
Summary
1.enzymes are catalysts
2.Lower activation energy EA
3.Mechanism of action …
4.Enzyme kinetics- Vmax, Km
5.Regulation of enzyme activity
- competitive, allosteric
phosphorylation