ENZYME KINETICS & REACTION RATE

Dr. Vivek A. Ojha, M.D.

 Summary:

[Link] kinetics and reaction rate.

[Link] of the rate of an enzymatic reaction.

1. The Michaelis-Menten Model.
2. Concepts and meaning of the constants Vmax, Km and Kcat.
3. Linearization of the Michaelis-Menten equation.

[Link] of cooperativity.
1. Kinetics deviating from the Michaelis-Menten model:
Enzymes with cooperativity
2. Co-operative models
3. Allosteric enzymes
 What is enzyme kinetics?

Why do you think it is important to

measure/understand enzyme kinetics?
1. Enzyme kinetics and reaction rate.
Enzyme kinetics is the study of the rate of biochemical reaction
catalysed by an enzyme. It helps us to understand enzymatic efficiency
and to determine the concentration of an enzyme in a solution or
biological sample.

The reaction rate of an enzyme is determined by the change in the

amount of substrate or product per unit time.

The determination of the rate of a biochemical reaction catalysed by

an enzyme can provide information about the presence of activators
or inhibitors, tissue damage, etc.

The study of how an enzyme responds to experimental parameters

can be used to: design inhibitors, design activators or modulate
activity.
 What is enzyme
kinetics?

Determining the rate of an

enzyme-controlled reaction
and studying how it changes
1. Enzyme kinetics and reaction rate.

How is enzyme activity measured?

V=-d[S]/dt = d[P]/dt
1. Enzyme kinetics and reaction rate.

How is enzyme activity measured?

V=-d[S]/dt = d[P]/dt
1 By measuring the disappearance of substrate or the emergence of product

2In the presence of very small quantities of enzyme, 10-8 and 10-12 M, quantities that are
known to be catalytic.
3 - Under optimal conditions of pH, temperature, presence of cofactors, etc.
4 – By saturating conditions of substrates are used
5 – By determining the initial reaction rate, V0
 2. Measurement of the rate of an enzymatic reaction.

reaction rate

Diagram source: [Link]

2. Measurement of the rate of an enzymatic reaction.

The rate of product formation is directly

proportional to the disappearance of the
substrate.

k1
E+S ES E+P
k2
k-1

To define the rate of the reaction we

need to define a steady state, where [ES]
is constant.
2. Measurement of the rate of an enzymatic reaction.
STEADY STATE in the enzyme reaction: WHEN THE CONCENTRATION OF THE COMPLEX
[ES] REMAINS CONSTANT.

V formation [ES]= V disappearance [ES].

The rate of change of [E] and [ES] with respect to time can
be considered 0, compared to the rate of change of [S] or
[P]. That is, d[ES]/dt=0.

ES E+P
k2

V=[ES]k2

Briggs and Haldane, 1925

2. Measurement of the rate of an enzymatic reaction.

The steady state in enzyme kinetics: assumption of application of the Michaelis-

Menten equation.
The ES complex is in a steady state when the entire enzyme is in ES form. In
this case, the rate of P formation is maximal for a given [S].

For substrate concentration the initial speed

V0 from [S4 ] is different.

The enzymes that behave in this way follow

the MICHAELIS-MENTEN model.

Biochemistry Stryer 7th edition

Michaelis Menten?
2. Measurement of the rate of an enzymatic reaction.
2.1. The Michaelis-Menten Model
The Michaelis-Menten equation governs enzymatic reactions.
Most enzymes exhibit Michaelis-Menten type kinetics, in which the graph of initial
velocity (V0) versus substrate concentration ([S]), is of the hyperbolic type.

Speed max=Vmax

V0 V= k2 [ES]
V0 /2

Km
[S]

The representation of V0 with respect to [S] is a

hyperbola
 Origin of the Michaelis-Menten equation
K1
E+S ES E+
K2
P K-1
In the steady state: Michaelis constant

V0 is maximum when [ES]=[Et ].

Initial Speed:

Michaelis-Menten equation (hyperbolic

curve):
2. Measurement of the rate of an enzymatic reaction.
2.1. The Michaelis-Menten Model
The Michaelis-Menten equation governs enzymatic reactions.
Most enzymes exhibit Michaelis-Menten type kinetics, in which the graph of initial
velocity (V0) versus substrate concentration ([S]), is of the hyperbolic type.

Speed max=Vmax
MICHAELIS-MENTEN
EQUATION

V0
V0 /2

Km
[S]

The representation of V0 with respect to [S] is a

hyperbola
2. Measurement of the rate of an enzymatic reaction.
2.2. Concepts and meaning of the constants Vmax, Km and Kcat

MEANING OF Vmax

It is the theoretical maximum speed

that is never reached and is constant.

It requires very high substrate

concentrations, and for all enzyme
molecules to be bound to substrate.

In the Michaelis-Menten equation

representation, the curve of V0 versus
[S] is asymptotically close.

From: Harvey, R., Ferrier, D. Bioquímica (Lippincot

Ilustrated Reviews) 7ª Edition, 2017
2. Measurement of the rate of an enzymatic reaction.
2.2. Concepts and meaning of the constants Vmax, Km and Kcat

MEANING OF Km

Substrate concentration at which the rate of Vmax/2 is

reached.

The Km may range in values from 10-1 to 10-6 M,

depending
on the enzyme.

The value of Km for an enzyme depends on the

particular substrate, and on other variables such as
temperature or pH.
Km establishes an approximate value for the intracellular
level of the substrate. The reaction rate is very sensitive From: Harvey, R., Ferrier, D.
to changes in [S] in the Km range Bioquímica (Lippincot Ilustrated
Reviews) 7th Edition, 2017
Km is usually related to an enzyme’s affinity for the
substrate.
2. Measurement of the rate of an enzymatic reaction.
2.2. Concepts and meaning of the constants Vmax, Km and Kcat

MEANING OF Km

Example: hexokinase and glucokinase have

different affinities for glucose.

Km of HEXOKINASE is very low at 0.1 mM,

this works for glycolytic purposes when
glucose concentration is low.
Km of GLUCOKINASE, 5 mM, will function at
high concentrations and for GLUCOGENIC
(storage) purposes.

Diagram source: [Link]

2. Measurement of the rate of an enzymatic reaction.
2.1. The Michaelis-Menten Model
The Michaelis-Menten equation governs enzymatic reactions.
Most enzymes exhibit Michaelis-Menten type kinetics, in which the graph of initial
velocity (V0) versus substrate concentration ([S]), is of the hyperbolic type.

Speed max=Vmax
MICHAELIS-MENTEN
EQUATION

V0
V0 /2

Km
[S]

The representation of V0 with respect to [S] is a

hyperbola
2. Measurement of the rate of an enzymatic reaction.
2.1. The Michaelis-Menten Model

1 Explanation of experimental data with the M-M

Speed max=Vmax
equation:

1.- [S] very low below the Km

Vmax x [S]
V0
V0 =
V0 /2 Km + [XS]
[S] is negligible versus Km and then V0 increases
linearly with [S]:
V0 = cte(Vmax/Km)x[S]
V x [S]
2.- [S] very HIGH and much HIGHER V0 = max
Km than Km XKm +
[S]
V0 = Vmax [S]
The representation of V0 with respect to [S] is a
3.- If V 0 = Vmax/2 then
hyperbola
Vmax x [S]
V0 = Vmax =Km + [S] Km=[S]
2. Measurement of the rate of an enzymatic reaction.
2.2. Concepts and meaning of Vmax, Km and Kcat constants

KCAT or TURNOVER NUMBER

Number of substrate molecules converted to product per enzyme

molecule per unit time, under substrate saturation conditions.
Kcat=Vmax/[E]T. The units are seconds-1.
2. Measurement of the rate of an enzymatic reaction.
2.3. Linearization of the Michaelis-Menten equation
Lineweaver-Burk plot

This is an inverse representation of V0 versus [S]:

1/V0 is plotted against 1/[S] and a straight line is

obtained. This is also called a double-reciprocal plot.

This is useful for calculating Km and Vmax more

accurately and the mechanism of action of enzyme
inhibitors.

The intercept on the x-axis is equal to -1/Km,

The y-axis intercept is equal to 1/Vmax. From: Harvey, R., Ferrier, D. Bioquímica
The slope m = Km/Vmax (Lippincot Ilustrated Reviews) 7th
Edition, 2017

What is the purpose of inverse representation?

When V0 is plotted against [S], it is not always possible to determine when Vmax has been
achieved due to the gradual upward slope of the curve at high substrate concentrations.
3. Phenomenon of cooperativity
3.1. Kinetics that do not behave according to the Michaelis-Menten
model: enzymes with cooperativity.

Some enzymes exhibit a sigmoid curve of V0 versus [S] or

sigmoid kinetics. This phenomenon indicates cooperativity in
the binding of the substrate to the active site. Enzyme following the
Michaelis-Menten model

Cooperativity: when the binding of one substrate molecule

to the enzyme affects the binding of subsequent substrate
molecules.
This behaviour is more common in multimeric enzymes with
several active sites. Enzyme with
cooperativity
Positive cooperativity: The binding of one substrate molecule
increases the affinity of the other active sites for the
substrate.
From: Harvey, R., Ferrier, D.
Negative cooperativity: The binding of one substrate Bioquímica (Lippincot
molecule decreases the affinity of the other active sites for Ilustrated Reviews) 7th
Edition, 2017
the substrate.
3. Phenomenon of cooperativity
3.1. Kinetics that do not behave according to the Michaelis-Menten
model: enzymes with cooperativity.

The phenomenon of cooperativity and sigmoidal behaviour in enzyme kinetics appears

in
multimeric enzymes that are also allosteric (a concept we will see later).
They have several active sites for substrate binding but these sites are not independent.

Enzyme with Affinity increases as sites are

cooperativity
occupied: Sigmoid curve

Michaelian
enzyme
The representation of V0 vs [S] has
sigmoidal behaviour.
Vmax/2 is referred to as K0.5
3. Phenomenon of cooperativity
3.2. Co-operative models
Cooperativity and allosteric effects can be explained by two models:
Monod-Wyman-Changeux concerted model. MWC
Koshland-Nemethy-Filmer sequential model. KNF

Both postulate that:

The enzyme subunits exist in one of two conformations: Tense
(T) conformation: lower affinity
Relaxed conformation (R): higher affinity to substrate

Explanation of kinetic behaviour

Biochemistry Stryer 7th edition

3. Phenomenon of cooperativity Tense (T) conformation: low-affinity
3.2. Co-operative models Relaxed conformation (R): high affinity

Concerted model: Monod-Wyman-Changeux

Upon binding of the first ligand, the

subunits change to the R state by
displacement of the equilibrium.

The subunits are changed

Sequential model: Koshland-Nemethy-Filmer sequentially

T R

[Link] Comprehensive Medicinal Chemistry III

2017, Pages 276-296
3. Phenomenon of cooperativity
3.3. Allosteric enzymes, Concept.

Allosteric enzymes are proteins with a quaternary structure, they have more than one
active and catalytic site, and their activity is regulated by allosteric modules.

Diagram source: [Link]

3. Phenomenon of cooperativity
3.3. Allosteric enzymes. Concept.

Allosteric enzymes can bind molecules other than substrates. They

are usually referred to as ligands or allosteric effects, which can
modulate enzyme activity.

ALLOSTERISM: a property of enzymes and other proteins that

undergoes a conformational change upon binding of a ligand such
that the affinity for that ligand or another ligand is affected.

ALLOSTERIC EFFECTOR: this is the ligand that induces the

conformational change – a binding modulator.
The activity of allosteric enzymes can be modulated by the binding of other ligands.
3. Phenomenon of cooperativity
3.3. Allosteric enzymes. Regulation of enzyme activity.

INHIBITOR

ACTIVATOR
3. Phenomenon of cooperativity
3.3. Allosteric enzymes. Regulation of enzyme activity.

Homotropic effectors
- The active site is also the regulatory site
- Binding at one site affects affinity at others (dependent)
If the homotropic effector facilitates binding, this is positive cooperativity
If it hinders binding, this is negative cooperativity
They are usually multimeric proteins with a binding site on each protein subunit.

HOMOTROPIC EFFECT ON EQUIVALENT SITES

The binding of O2 favours

binding at the other sites.
conformational change
that facilitates the
binding of the second
molecule
 3. Phenomenon of cooperativity
3.3. Allosteric enzymes. Regulation of enzyme activity.
Heterotropic effectors
- This is another molecule that alters the affinity of enzyme-substrate binding
- Can inhibit or activate binding
- They bind to a regulatory site, which is different from the active site of the enzyme.
If the heterotropic effector facilitates binding, this is positive cooperativity.
If it hinders binding, this is negative cooperativity.

NEGATIVE HETEROTROPIC EFFECT POSITIVE HETEROTROPIC

EFFECT

Molecular Biology of the Cell, 6th edition, Bruce Alberts

NEGATIVE HETEROTROPIC EFFECT POSITIVE HETEROTROPIC EFFECT

Molecular Biology of the Cell, 6th edition, Bruce Alberts

3. Phenomenon of cooperativity
3.3. Allosteric enzymes. Examples.

Glycogen phosphorylase: key enzyme in glycogenolysis

It has an allosteric regulation system that responds to a low energy load.

Glucose and ATP: favourable energy

conditions shift enzyme equilibrium
towards the tense form.
Negative allosteric effectors/inhibitors. TENSE FORM RELAXED FORM
+
AMP
AMP: poor energy conditions, shifts the
balance to the active, relaxed form.
Positive allosteric effect activator. GLUCOSE
INACTIVE ATP ACTIVE

-
3. Phenomenon of cooperativity
3.3. Allosteric enzymes. Examples.

Phosphofructokinase 1 (PFK1) – involved in glycolysis

This glycolytic enzyme is allosterically inhibited by citrate, which is not a substrate for the
enzyme. This is an example of feedback inhibition, in this case by citrate.

PFK1
Fructose-6-Pi + ATP F-1,6-bisPi + ADP
+

Fructose-6-Pi PYRUVATE PYRUVATE CITRATE

-
3. Phenomenon of cooperativity
3.3. Allosteric enzymes. Examples.
Aspartate transcarbamoylase (ATCase): an important enzyme in the synthesis
of pyrimidine nucleotides

ATCase catalyses the regulated step of the pathway, is inhibited by the end product CTP, and is
activated by ATP (purines).

ALLOSTERIC SUBUNIT: YELLOW,

linking the effectors ATP
CATALYTIC SUBUNIT:
ATP PINK

CTP ATP

Mechanism of CTP inhibition: Mechanism of ATP activation:

Stabilises T-shapes Stabilises R-shapes
3. Phenomenon of cooperativity
3.3. Allosteric enzymes. Examples.
Aspartate transcarbamoylase (ATCase) – an enzyme important in the
synthesis of pyrimidine nucleotides

Lower activity and less sensitive curve to Higher activity and more sensitive
substrate concentration curve to substrate concentration
4. Types of protein-ligand interactions

P-L interactions:
- are specific
- occur with weak bonds: van der Waals, hydrogen bridges, hydrophobic and ionic bonds

The functionality of proteins depends on dynamic properties:

1. Flexibility: ability to modulate a large number of weak links.
2. Conformational changes: small variations of the tertiary structure that have an effect on the
functionality/activity of proteins
[Link]: which may be reversible interactions with ligands (inhibitors,
activators, substrates, etc.) or protein-protein interactions.

The functions of ligand-binding

proteins are affected by ligand
binding.
THANKS