REGULATION OF ENZYME ACTIVITY
HOW ENZYMES WORK ?
 Enzyme-catalyzed reactions are characterized by the formation of a complex between
substrate and enzyme (an ES complex).
 Substrates bind to a pocket on the enzyme called the ACTIVE SITE
 The function of enzymes is to lower the activation energy and in that way enhance the
reaction rate.
REGULATORY ENZYMES
 In the body, thousands of diverse enzymes are regulated to
fulfill their individual functions without waste of dietary
components. Thus, with changes in our physiologic state, time
of eating, environment, diet or age, the reaction rates of some
enzymes must increase and others decrease
 Regulatory enzymes exhibit increased or decreased catalytic
activity in response to certain signals.
 Adjustments in the rate of reactions catalyzed by regulatory
enzymes and ,therefore, in the rate of the entire metabolic
pathway, allow the cell to meet changing needs for energy and
for biomolecules required in growth and repair.
ENZYME KINETICS AS AN APPROACH
TO UNDERSTANDING MECHANISM
The central approach to studying the mechanism of an enzyme-catalyzed reaction is to determine
the rate of the reaction, a discipline known as enzyme kinetics
ENZYME KINETICS
 Most enzymes have certain kinetic
properties in common.
 When substrate is added to an enzyme,
the reaction rapidly achieves a STEADY
STATE: [ES] is constant, the ES formation
equals the rate of ES breakdown
 As [S] increases, the steady-state activity
of a fixed concentration of enzyme
increases in a HYPERBOLIC FASHION to
approach a characteristic maximum rate,
Vmax (essentially all the enzyme has
formed a complex with substrate).
SUBSTRATE CONCENTRATION AFFECTS THE RATE
OF ENZYME-CATALYZED REACTIONS




A key factor affecting the rate of enzyme catalyzed reaction is [S].
At low [S], most of the enzyme is in free form (E); the rate is proportional to [S].
The equilibrium is pushed toward formation of more ES as [S] increases.
The maximum reaction rate (Vmax) is observed when virtually all the enzyme is present as the ES
complex. Under these conditions, the enzyme is “saturated” with its substrate, so that further
increases in [S] have no effect on the reaction rate.
MICHAELIS-MENTEN MODEL OF ENZYME KINETICS
 The Michaelis-Menten equation relates initial
reaction velocity to [S] and Vmax through
constant Km.
v
V max  S 
Km  S 
 The Michaelis-Menten kinetics is called the
steady-state kinetics.
 The [S] that results in a reaction rate equal to
one-half of Vmax is the Michaelis constant - Km
(characteristic for each enzyme acting on a given
substrate)
 Vmax of the enzyme is the maximal velocity that
can be achieved at an infinite [S]
MICHAELIS-MENTEN EQUATION
* This equation is derived for a reaction in which a single substrate, S is converted to a single product, P
 k1 - the rate constant of ES formation
 k2 The rate constant of ES complex dissociation
 K3 the rate constant of ES conversion to P
LINEWEAVER-BURK PLOT
HEXOKINASE ISOZYMES HAVE DIFFERENT KM VALUES
FOR GLUCOSE
 Isozymes of hexokinase are a good example of
the significance of the Km of an enzyme for its
substrate
 Hexokinase I, the isozyme in erythrocytes, has a
Km for glucose of 0.05 mM.
 Glucokinase (found in the liver and pancreas),
has a much higher Km of 5-6 mM.
At the low Km of the erythrocyte hexokinase,
blood glucose could fall drastically below its
normal fasting level of approximately 5 mM,
and the red blood cell could still phosphorylate
glucose at rates near Vmax.
The high Km of hepatic glucokinase thus
promotes the storage of glucose, as liver
glycogen or as fat, but only when glucose is in
excess supply.
KINETIC PARAMETERS ARE USED TO COMPARE
ENZYME ACTIVITIES
 Km can vary greatly from enzyme to enzyme, and even for different substrates of the same
enzyme.
 Km depends on specific aspects of the reaction mechanism (number and relative rates of the
individual steps).
 More general rate constant, Kcat describes the limiting rate of any enzyme-catalyzed reaction at
saturation. If the reaction has several steps and one is clearly rate limiting, Kcat is equivalent to
the rate constant for that limiting step.
Kcat – TURNOVER NUMBER
 The constant Kcat is equivalent to the number of substrate molecules converted to product in a
given unit of time on a single enzyme molecule when the enzyme is saturated with substrate.
MANY ENZYMES CATALYZE REACTIONS
WITH TWO OR MORE SUBSTRATES
A. Both substrates are bound to the
enzyme at some point in the course
of the reaction, forming a
noncovalent ternary complex; the
substrates bind in a random sequence
or in a specific order.
B. The first substrate is converted to
product and dissociates before the
second substrate binds, so no ternary
complex is formed. An example of this
is the Ping-Pong, or doubledisplacement, mechanism.
MANY ENZYMES CATALYZE REACTIONS
WITH TWO OR MORE SUBSTRATES
NONCOVALENT TERNARY COMPLEX
TRANSAMINATION REACTION – PING-PONG MECHANISM
THE MECHANISMS FOR REGULATION OF ENZYMES
1. Regulation by compounds that bind in the active site
(dependence of velocity on substrate concentration, (ir)reversible inhibitors)
2. Regulation by changing the conformation of the active site
(allosteric regulators, covalent modification, protein–protein interactions, zymogen cleavage)
3. Regulation by changing the concentration of enzyme
(enzyme synthesis and degradation)
ENZYMES ARE SUBJECT TO REVERSIBLE
OR IRREVERSIBLE INHIBITION
REVERSIBLE INHIBITION
- Competitive inhibition  Competitive inhibition - a competitive inhibitor competes with the substrate in the active site of an
enzyme.
 While the inhibitor (I) occupies the active site, it prevents binding of the substrate to the enzyme.
REVERSIBLE INHIBITION
- Competitive inhibition -

Many competitive inhibitors are compounds
that resemble the substrate and combine with
the enzyme to form an EI complex, BUT
WITHOUT LEADING TO CATALYSIS.

This affects the Km, however with the absence
of an effect on Vmax and is easily revealed in a
double reciprocal plot.
 A medical therapy based on competition at the active site is used to treat
patients who have ingested methanol, a solvent found in gas-line
antifreeze. The liver enzyme alcohol dehydrogenase converts methanol
to formaldehyde, which is damaging to many tissues (e.g. blindness).
 Ethanol competes effectively with methanol ,as an alternative substrate
for alcohol dehydrogenase. The effect of ethanol is much like that of a
competitive inhibitor, with the distinction that ethanol is also a substrate
for alcohol dehydrogenase and its concentration will decrease overtime
as the enzyme converts it to acetaldehyde.
 The therapy for methanol poisoning is slow intravenous infusion of
ethanol, at a rate that maintains a controlled concentration in the
bloodstream for several hours.
REVERSIBLE INHIBITION
- Uncompetitive and noncompetitive inhibition  These two types of reversible inhibition, uncompetitive and noncompetitive/mixed, although
often defined in terms of one substrate enzymes, are in practice observed only with enzymes having
two or more substrates.
 An UNCOMPETITIVE INHIBITOR binds at a site distinct from the substrates active site and, unlike a
competitive inhibitor, binds only to the ES complex.
 A NONCOMPETITIVE (MIXED) INHIBITOR also binds to a site distinct from the substrates active
site, but it binds to either E or ES.
REVERSIBLE INHIBITION
- Noncompetitive inhibition 
If an inhibitor does not compete with a substrate for its binding site, the inhibitor is either a
noncompetitive or uncompetitive inhibitor with respect to that particular substrate.
 Noncompetitive inhibition in a multisubstrate reaction
(substrates A and B):
1. An inhibitor (NI) that is a structural analog of substrate
B would fit into substrate B’s binding site, but the
inhibitor would be a noncompetitive with regard to the
substrate A.
2. An increase of A will not prevent the inhibitor from
binding to substrate B’s binding site.
3. The inhibitor lowers the concentration of the active
enzyme and therefore changes the Vmax.
REVERSIBLE INHIBITION
- Noncompetitive inhibition -
 If the inhibitor has absolutely no effect on
the binding of substrate A, it will not change
the Km for A (A PURE NONCOMPETITIVE
INHIBITOR), however it lowers the Vmax.
 Some inhibitors, such as metals, might not
bind at either substrate recognition site. In
this case, the inhibitor would be
noncompetitive with respect to both
substrates.
REVERSIBLE INHIBITION
-Uncompetitive inhibition •
•
An inhibitor that is uncompetitive with respect to a substrate will bind ONLY TO ENZYME
CONTAINING THAT SUBSTRATE.
The uncompetitive inhibitor would decrease the Vmax of the enzyme and affect its Km.
IRREVERSIBLE INHIBITION

The irreversible inhibitors bind covalently with or destroy a functional group on an enzyme,
or form a particularly stable non-covalent association

The suicide inactivators are a special type of irreversible inhibitors, which 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

They are converted to a highly reactive compound that combines irreversibly with the
enzyme

These compounds are also called mechanism-based inactivators
IRREVERSIBLE INHIBITION
- HEAVY METAL POISONING -
IRREVERSIBLE INHIBITION
- PENICILLIN -
The antibiotic penicillin is a
TRANSITION STATE ANALOG that binds
very tightly to glycopeptidyl
transferase, an enzyme required by
bacteria for synthesis of the cell wall.
Glycopeptidyl transferase catalyzes a
partial reaction with penicillin
that covalently attaches penicillin to its
own active site serine.
Penicillin thus undergoes partial
reaction and acts as irreversible
inhibitor in the active site or “suicide
inhibitor”.
Key points:

In Michaelis-Menten (steady-state kinetics) Km and Vmax have different values for different
enzymes.

The limiting rate of an enzyme-catalyzed reaction at saturation is best described by the constant
Kcat, the turnover number.

The Michaelis-Menten equation is also applicable to bisubstrate reactions, which occur by ternarycomplex or Ping-Pong (double-displacement) mechanism.

Reversible inhibition of an enzyme might be competitive, uncompetitive or noncompetitive.
1.
Competitive inhibitors compete with substrate by binding reversibly to the active site, but
they are not transformed by the enzyme.
2.
Uncompetitive inhibitors bind only to the ES complex, at a site distinct from the active site.
3.
Noncompetitive inhibitors bind to either E or ES, at a site distinct from the substrates binding
site.

In irreversible inhibition an inhibitor binds permanently to the active site by forming a covalent
bond or a very stable noncovalent interaction.
ALLOSTERIC ENZYMES UNDERGO CONFORMATIONAL CHANGES
IN RESPONSE TO MODULATOR BINDING
 The activity of allosteric enzymes is
adjusted by reversible binding of a specific
modulator to a regulatory site
 Modulators may be the substrate itself or
some other metabolite
 The effect of modulator might be inhibitory
or stimulatory
 The behavior of allosteric enzymes reflects
cooperative interactions among enzyme
subunits
CONFORMATIONAL CHANGES IN ALLOSTERIC ENZYMES
 Allosteric activators and inhibitors - ALLOSTERIC EFFECTORS are:

compounds that bind to the ALLOSTERIC SITE (a site separate from the catalytic site)

cause a conformational change that affects the affinity of the enzyme for the substrate
 Usually an allosteric enzyme has multiple interacting subunits that can exist in active and
inactive conformations
 Allosteric effector promotes conversion from one conformation to another
ALLOSTERIC ACTIVATORS AND INHIBITORS
 ALLOSTERIC ACTIVATOR:
1. changes the conformation of the active site in a way that increases the affinity of the enzyme
for the substrate
2. is more tightly to the high-affinity, relaxed (R) state, than the tense (T) state
3.
the activators increase the amount of enzyme in the active state, by facilitating substrate
binding in their own and other subunits of the allosteric enzyme
 ALLOSTERIC INHIBITORS:
1. bind more tightly to the T state
2. changes the conformation of the active site in a way that decreases the affinity of the enzyme
for the substrate
3. either substrate or activator concentration must be increased to overcome the effects of the
allosteric inhibitor
COOPERATIVITY IN SUBSTRATE BINDING TO
ALLOSTERIC ENZYMES
 Allosteric enzymes usually contain
two or more subunits and exhibit
positive cooperativity
 The binding of substrate to one
subunit facilitates the binding of
substrate to other subunit(s)
COOPERATIVITY IN SUBSTRATE BINDING TO
ALLOSTERIC ENZYMES
 The first substrate molecule has
difficulty in binding to the enzyme
because all of the subunits are in the
conformation with a low affinity for
substrate – the tense or taut “T”
conformation.
 The first substrate molecule
binding changes its own subunit and
at least one adjacent subunit to the
high-affinity conformation - the
relaxed “R” state
THE KINETIC PROPERTIES OF ALLOSTERIC ENZYMES DIFFER
FROM MICHAELIS-MENTEN BEHAVIOR
 Sigmoid kinetic behavior generally reflects cooperative interactions between protein subunits.
 An activator may cause the curve to become more nearly hyperbolic, with a decrease in K0.5 , but
no change in Vmax.
 A negative modulator (an inhibitor) may produce a more sigmoid substrate-saturation curve,
with an increase in K0.5
PROTEIN KINASE A
 When the regulatory subunits (R) of protein kinase A bind the
allosteric activator, cAMP, they dissociate from the enzyme,
thereby releasing active catalytic subunits (C).
 Some PK are tightly bound to a single protein, regulating its
activity. Other protein kinases and protein phosphatases
simultaneously regulate a number of rate-limiting enzymes
to achieve a coordinated response.
 PKA, a serine/threonine protein kinase, phosphorylates a
number of enzymes that regulate different metabolic
pathways.
 Protein kinase A provides a means for hormones to control
metabolic pathways:
 Adrenaline and many other hormones increase the
intracellular concentration of the allosteric regulator
cAMP - a hormonal second messenger.
 cAMP binds to regulatory subunits of PKA, which
dissociate and release the activated catalytic subunits.
IN MANY PATHWAYS A RATE-LIMITING STEP IS CATALYZED BY
AN ALLOSTERIC ENZYME
 The activities of metabolic pathways in the cells are regulated by control of the activities of
certain enzymes
 In feed-back inhibition, the end product of a pathway inhibits the first, rate-limiting, enzyme of
that pathway
SIMPLE PRODUCT INHIBITION
 Simple product inhibition - a decrease in the rate of an enzyme caused by the accumulation of its
own product, plays an important role in metabolic pathway, since it prevents one enzyme in a
sequence of reactions from generating a product faster than it can be used by the next enzyme in
that sequence
PRODUCT INHIBITION OF HEXOKINASE
•
Product inhibition of hexokinase by glucose 6-P
conserves blood glucose for tissues needing it.
•
Tissues take up glucose from the blood and
phosphorylate it to glucose 6-P, which can then
enter a number of different pathways.
•
As these pathways become more active, glucose 6-P
concentration decreases and the rate of hexokinase
increases.
•
When these pathways are less active, glucose 6phosphate concentration increases, hexokinase is
inhibited and glucose remains in the blood for other
tissues.
Ann O’Rexia, a 23-year old woman, is being treated for anorexia nervosa. She has been gaining
weight slowly. Her blood glucose is still below normal (72 mg/dL compared to a normal range of
80-100 mg/dL). She complains to her physician that she feels tired when she jogs and she is
concerned that the “extra weight” she has gained is slowing her down.
 One of the fuels used by skeletal muscles for jogging is
glucose. Glucose 6-P is metabolized in glycolysis to
generate ATP. This pathway is feed-back regulated, so
that as her muscles use more ATP, the rate of glycolysis
will increase to generate more ATP.
 When she is resting, her muscles and liver will convert
glucose 6-P to glycogen. Glycogen synthesis is feedforward regulated by the supply of glucose and by
insulin and other hormones.
 Glycogenolysis (glycogen degradation) is activated
during exercise to supply additional glucose 6-P for
glycolysis.
 Unless Ann consumes sufficient calories (glucose), her
glycogen storages will not be replenished after exercise
and she will tire easily.
Key points:
 In many pathways a rate-limiting step is catalyzed by an allosteric enzyme
 The activity of allosteric enzymes is adjusted by reversible binding of a specific
modulator to a regulatory site (substrate or some other metabolite)
 An allosteric enzyme has multiple interacting subunits that can exist in active (R) and
inactive (T) conformations, while the conversion is regulated by allosteric effector
(activator or inhibitor)
 Allosteric enzymes usually contain two or more subunits and exhibit positive
cooperativity
 Sigmoid kinetic behavior generally reflects cooperative interactions between protein
subunits
 In feed-back inhibition, the end product of a pathway inhibits the first enzyme of
that pathway
SOME REGULATORY ENZYMES UNDERGO
REVERSIBLE COVALENT MODIFICATION
 Modifying groups include phosphoryl, adenylyl, uridylyl, methyl and adenosine diphosphate
ribosyl groups .
 These groups are generally linked to and removed from the regulatory enzyme by separate
enzymes
PHOSPHORYL GROUPS AFFECT STRUCTURE AND CATALYTIC
ACTIVITY OF ENZYMES
 The addition of a phosphoryl group to a Ser, Thr or Tyr residue introduces a bulky, charged
group into a region that was only moderately polar.
 The oxygen atoms of a phosphoryl group can hydrogen-bond with one or several groups in a
protein.
 When the modified side chain is located in a region of the protein critical to its threedimensional structure, phosphorylation can have dramatic effects on protein conformation and
thus on substrate binding and catalysis.
SOME REGULATORY ENZYMES USE SEVERAL
REGULATORY MECHANISMS
- REGULATION OF GLYCOGEN PHOSPHORYLASE ACTIVITY BY COVALENT AND NONCOVALENT MODIFICATIONS -
 Glycogen phosphorylase exists in 2 forms: phosphorylase a and phosphorylase b
 In the more active form of the enzyme, phosphorylase a, specific Ser residues, one on each
subunit, are phosphorylated
 Phosphorylase a is converted to the less active, phosphorylase b , by dephosphorylation, catalyzed
by phosphorylase phosphatase
 Phosphorylase b can be reconverted (reactivated) to phosphorylase a by the action of
phosphorylase kinase.
When Ann O’Rexia begins to jog, AMP activates
her muscle glycogen phosphorylase, which
degrades glycogen to glucose 1-P. This compound
is converted to glucose 6-P, which feeds into the
glycolytic pathway to generate ATP for muscle
contraction.
As she continues to jog, her adrenaline (epinephrine) levels rise, producing the signal that activates
glycogen phosphorylase kinase. This enzyme phosphorylates glycogen phosphorylase, causing it to
become even more active than with AMP alone
 When Ann O’Rexia jogs, the increased use
of ATP for muscle contraction results in an
increase of AMP, which allosterically
activates glycogen phosphorylase, the
rate-limiting enzyme of glycogenolysis, but
also allosteric enzyme
phosphofructokinase- 1, the rate-limiting
enzyme of glycolysis
 This is an example of feed-back regulation
by the ATP/AMP ratio
 Unfortunately, her low caloric
consumption has not allowed feedforward activation of the rate-limiting
enzymes in her fuel storage pathways,
therefore she has very low glycogen
stores.
 Consequently, she has inadequate fuel
stores to supply the increased energy
demands of exercise and, thus, feels tired
SOME ENZYMES ARE REGULATED BY PROTEOLYTIC CLEAVAGE OF
AN ENZYME PRECURSOR
 Zymogens are enzyme precursors or inactive forms of enzymes
 Zymogens are activated by proteolytic cleavage
REMAINING POLYPEPTIDE CHAINS ARE
CONNECTED BY DISULFIDE BONDS
REGULATION THROUGH CHANGES IN AMOUNT OF ENZYME
A. Regulation of Enzyme Synthesis
 Protein synthesis begins with the process of gene transcription, transcribing the genetic
code for that protein from DNA into mRNA.
 Generally the rate of enzyme synthesis is regulated by increasing or decreasing the rate
of gene transcription, processes generally referred to as induction (increase) and
repression (decrease).
 The rate of enzyme synthesis is sometimes regulated through stabilization of the mRNA.
 Regulation by means of induction/repression of enzyme synthesis is usually slow in the
human, occurring over hours to days.
B. Regulation of Protein Degradation
 Although all proteins in the cell can be degraded with a characteristic half-life within
lysosomes, protein degradation via two specialized systems, proteosomes and caspases,
is highly selective and regulated.
REGULATION OF THE RATE
OF SYNTHESIS OF
HMG-CO REDUCTASE
Key points:

Regulatory enzymes are modulated by covalent modification of a specific functional
group necessary for its activity.

The phosphorylation of specific amino acid residues is a particularly common way to
regulate enzyme activity.

Many proteolytic enzymes are synthesized as inactive precursors called zymogens, which
are activated by cleavage of small peptide fragments.

Enzymes at important metabolic intersections may be regulated by several regulatory
mechanisms and complex combinations of effectors, allowing coordination of the
activities of interconnected pathways