EDO UNIVERSITY IYAMHO

Department of Biochemistry

BCH 411 Advanced Enzymology

Instructor I: Dr. Anthony M. UGBENYEN, email: [email protected]
Instructor II: Dr. Sheu K. RAHAMON, email: [email protected]
Lectures: Tuesday, 9 am – 11am, LT3, Phone: 08138071549, 08067420607
Office hours: Tuesday, 12 pm to 1 pm, Office: Ground floor &1st floor, New College
Building

General overview of lecture: This course is intended to build on the basic knowledge of
enzymology acquired by the student in 300 level. The course covers advanced topics in
Enzymology such as enzyme kinetics and enzyme isolation, purification and characterization.

Prerequisites: Students should be familiar with basic knowledge of enzymology such as

properties, nomenclature, basic enzyme kinetics, relevance of kinetic parameters and
experimental determination of kinetic parameters among others.

Learning outcomes:At the end of the lectures, students should be able to:

1. Demonstrate improved understanding of enzyme kinetics

2. Discuss the chemistry of enzyme catalysis
3. Discuss catalysis in multi-enzyme system
4. Demonstrate an understanding of enzyme isolation, purification, characterization and
reconstitution

Assignments: We expect to have at least 3 individual homework and 1 oral presentation

throughout the course in addition to a Mid-Term Test and a Final Examination. Home works
are due at the beginning of the class on the due date. Home works are organized and
structured as preparation for the midterm and final examination, and are meant to be a
studying material for both examinations.

Grading: We will assign 10% of this class grade to homework, 10% to oral presentation and
10% for the mid-term test. The final examination, which will be comprehensive, shall be
70%.

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Textbook: The recommended textbook for this class are as stated:
Title: Biochemistry: The Chemical Reactions of Living Cells (Volumes 1 & 2)
Authors: David E. Metzler
Publisher: Elsevier Academic Press, 2nd edition

Title: Enzyme Kinetics: Theory and Practice in: Plant Metabolic Networks (Schwender (ed.)
Authors: Alistair Rogers and Yves Gibon.
Publisher: Springer Science+Business Media, LLC 2009
DOI 10.1007/978-0-387-78745-9_4

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 ENZYME KINETICS

Enzyme kinetics deals with measuring the speed (reaction rate) of an enzymatic reaction. Its
goal is to establish a “rate equation” which describes the velocity of a reaction interms of
kinetic constants and other parameters which are measured experimentally and described
mathematically.

In measuring velocity, an enzyme catalysed reaction is started at a definite time by mixing

two or more substrates (reactants) together rapidly. Usually, the reaction is kept at a precise
constant temperature and pH, and the concentration of the substrate(s) or product(s)is/are
measured aftera fixed time interval or at different intervals. The disappearance of the
substrate(s) and the appearance of the product(s) with time can then be shown as a progress
curve (as shown below).

Figure 1: Progress curve showing increased [P] or decreased [S]

For many steps in metabolism, enzyme kinetic properties have been determined, and this
information has been collected and organized in publicly available online databases
(www.brenda.uni-koeln.de).

VELOCITY OF AN ENZYMATIC REACTION

The velocity (v) of an enzymatic reaction is defined as the rate at which a substrate disappears
or at whicha product is formed. The two can be represented mathematically as shown below.

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The units of velocity is moles per litre per second (M s–1) but traditionally in enzymology, it
is moles per litre per minute.

STEADY STATE AND TRANSIENT STATE ENZYME KINETICS

When an enzyme is mixed with an excess of substrate, there exists an initial short period of
time (few 100 microseconds) during which intermediates leading to the formation of product
gradually build up. This state is called pre-steady state or transient state. Due to the rapid
nature of this state, special technique is usually required to study its kinetics.

After this initial state (i.e. transient state), a steady state is reached where reaction rates and
concentration of intermediate change relatively slowly with time.Measurement of the
progress of reaction during the steady state is characterised by reduction in [S] with an
equivalent increase in [P] as shown in Figure 1.

Steady state kinetics is based mainly on Michaelis-Menten equation which rests on 2

assumptions:

1. Equilibrium assumption
The rate of formation of enzyme substrate (ES) complex from free enzyme and substrate is
equal to the rate of dissociation of ES complex to free enzyme and substrate i.e.The rate of

formation of ES = Rate of its disappearance.

In this state, backward and forward reactions are at equilibrium.

2. Steady state assumption

The concentration of ES complex remains relatively constant over a period of rate
measurement until S is almost totally exhausted. In this state, the rate of synthesis of ES
complex is equal to the rate of its breakdown.

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REACTION RATES AND REACTION ORDER
Reactions can be of different types. Some of these types include First-Order Irreversible
Reaction, First-Order Reversible Reaction and Second-Order Reaction.

First-order reactions

First-order kinetics is observed for unimolecular processes in which a molecule of A

(reactant) is converted into product P in a given time interval with a probability that does not
depend on interaction with another molecule e.g. radioactive decay.

In first-order reaction, the rate depends on the first power of the concentration i.e. the rate of
decrease of the concentration of a given reactant [A] is experimentally directlyproportional to
the concentration of that reactant atany given time.

A. First-Order Irreversible Reaction

The simplest possible reaction is the irreversible conversion of substance A to product

P Eqn 1
The arrow in the reaction above signifies that the equilibrium lies far to the right, and
the reverse reaction is infinitesimallysmall. As discussed earlier, the reaction rate or
velocity (v) of the reaction can be defined in terms of the time (t)-dependent
production of product P. Alternatively, the reaction rate or v can also be defined in
terms of the time-dependent consumption of substance A, since formation of P
involves the loss of A.
First-Order Irreversible Reaction is mathematically expressed as:

Eqn 2 where [A] and [P] are the concentrations of

the substance and product, respectively.
As A is transformed to P, there is less of A to undergo the transformation, and
therefore the concentration of A will decrease exponentially with time.

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 Time
Figure 2: First-order reaction showing the decrease of A over time

The rate constant (k1) of this reaction is proportional to the concentration of A and has the
unit s−1.

Integration of Eqn 2 fromtime zero (t0) to time t gives Eqn 3

OR

OR (equation of exponential decay) where [A]0 is the

starting concentration at t0
Whenln[A] is plotted against t, a first-order reaction will yield a straight line, where
the gradient is equal to –k1 (Figure 3).

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 Figure 3: First-order reaction showing the decrease of ln[A] over time

B. First-Order Reversible Reaction

In Biochemistry, few reactions are as simple as the first-order reaction. In most cases,

reactions are reversible and equilibrium does not lie far to one side. Eqn 4

Therefore, the corresponding rate equation is given as

Eqn 5 where where k1 and k–1 are the rate

constants for the first-order, forward and reverse, reactions respectively.

When the rates of the forward and reverse reactions are equal, consumption of A will stop
and the overall reaction rate is zero, i.e., a stateof equilibrium is attained.

Eqn 6 where [A]eq and [P]eq are the substrate

concentrations at equilibrium.

It should be noted that in catalyzed reactions, the position of equilibrium is not altered by the
presence of an enzyme but the enzyme increases the rate at which equilibrium is attained.

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When the forward and reverse reactions are both first order, the equilibrium constant (Keq) is
equal to the ratio of the rate constants for the forward and reverse reactions. For a reaction to
precede in the direction of product (P) formation, the equilibrium constant must be large.

Eqn 7

PSEUDO-FIRST ORDER REACTION

A reaction is considered pseudo-first order; when substance A actually reacts with a second
molecule such as water, which is present in such excess that its concentration does not change
during the experiment. Consequently, the velocity is apparently proportional only to [A].

SECOND-ORDER REACTION
For a chemical reaction to occur between two molecules, A and B, they must meet and
collide.The velocity of a second-order process is characterized by a bimolecular rate constant
and is proportional to the product of the concentrations of A and B.
Whenever two reactants come together to form a product, the reaction is considered second

order, e.g., Eqn 8

In addition to being reversible, most reactions are second order or greater in their complexity.
The rate of reaction in Eqn 8 is proportional to the consumption of A and B and to the
formation of P. A reaction is described as second order because the rate is proportional to the
second power of the concentration.
The rate constant k1 has the unit s–1 M–1

Eqn 9
Integration of Eqn 9 yields an equation where t is dependent on the two variables,
A and B. To solve this equation, an assumption that either A or B is constant must be made.
This is achieved experimentally by using a concentration of B that is far
in excess of requirements such that only a tiny fraction of B is consumed during the reaction
and therefore the concentration can be assumed not to change. The reaction can then be
considered as pseudo-first order.

Eqn 10

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On the other hand, if the concentration of both A and B at time zero are the same, i.e., [A0] =
[B0], then Eqn 10 can be simply written as:

Eqn 11

THE MICHAELIS–MENTEN EQUATION

The Michaelis–Menten equation was derived by Michaelis and Menten but was further
developed by Briggs and Haldane. The equation is fundamentally importantto enzyme
kinetics and it is characterized by two constants: theMichaelis–Menten constant (Km) and the
indirectly obtained catalytic constant, kcat.

Derivation of M-M Equation

As you will recall, a step-by-step derivation of the M-M equation was done in 300 level
(BCH 312). However, a simple revision is provided here using the simple conversion of
substrate (A) into product (P) catalyzed by the enzyme (E).

As explained in induced-fit hypothesis, the first step is substrate binding and the second step
is the catalytic step.

Eqn 12

Referring to Eqn 2, , formation of P can be

defined in terms of the dissociation rate (k2) of the EA complex, commonly denoted as kcat,
and the concentration of the enzyme–substrate complex ([EA])

Eqn 13

Assumption

The dissociation rate (kcat in Eqn 13 or k2 in Eqn 12) of the EA complex is assumed to be
slow compared to association (k1) and redissociation (k-1) reactions and that the reverse
reaction (P→A) is negligible.

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Change in substrate (A), product (P), free enzyme (E), enzyme–substrate complex (EA), and
total enzyme (Et) concentration over time

Using a simple reaction such as Eqn12 , the

interaction between A and E, formation of EA and the eventual formation of P can be divided
into 3 distinct phases.

The first phase is a very brief initial period during which the concentration of enzyme–
substrate complex [EA] reaches a steady state in which consumption and formation of EA
complex are balanced.

During the second phase, the [EA] remains almost constant for a considerable time; this
period is known as the steady state, this is the condition described by Michaelis–Menten
equation.

The third phase of the reaction is characterized by substrate depletion in which [EA]
gradually falls as Ais consumed, this facilitates a rise in the concentration of the free enzyme
since there is no A to bind to.

Figure 4: Change in A, P, free enzyme (E), EA, and total enzyme (Et) concentration over time

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At steady state, [EA] is stable, i.e., Eqn 14

Therefore, the association reaction (formation of ES complex) and the sum of the
redissociation and dissociation reactions (the breakdown of ES complex) are equal.

Eqn 15

Rearrangement of Eqn 15 by making EA the subject of the formula yields

Eqn 16

The three rate constants can now be combined as one term. This new constant, Km, is known
as the Michaelis–Menten constant

Eqn 17

Rewriting Eqn 16, you have

Eqn 18

But the amount of free enzyme (E) and enzyme that is bound to the substrate (EA) variesover
the course of a reaction, while the total amount of enzyme (Et) is constant

Eqn 19

Substituting Eqn 19 into Eqn 18, you have

Eqn 20

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Rearrangement of Eqn 20 yields

Eqn 21

Substituting into Eqn13 , you have

Eqn 22

But the maximum possible reaction rate (vmax) would be achieved when all the available
enzyme is bound to A and involved in catalysis, i.e.,

Eqn 23

Substituting Eqn 23 into Eqn 13 under conditions of saturating [A] yields

Eqn 24

Substituting Eqn 24 into Eqn 22 yields the Michaelis–Menten equation

Eqn 25

Key Parameters of the Michaelis–Menten Equation

There are four key parameters of the M-M equation. These include Km, kcat, Km/kcat ratio and
vmax.
A. Km (mol.l−1)
The Km for a given enzyme is constant provided a stable pH, temperature, and redox
state is assumed. Kmprovides an indication of the binding strength of an enzyme to its
substrate.
M-M kinetics assumes that kcat is very low when compared to k1 and k–1. Therefore, a
high Km indicates that the redissociation rate (k–1) is markedly greater than the

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 association rate and that the enzyme binds the substrate weakly. Conversely, a low Km
indicates a higher affinity for the substrate.
It must be noted however, that a large Km could also be the result of very large kcat
(see Eqn 17) therefore, caution must be taken when using Km as a proxy for the
dissociation equilibrium constant of the EA complex.

Eqn 17
–1
B. kcat (s )
kcat, is a measure of the maximum catalytic production of P under saturating substrate
conditions per unit time per unit enzyme. It is also referred to as the turnover number
of the enzyme.
The larger the value of kcat, the more rapidly catalyticevents occur. Values of kcatvary
markedly from one enzyme to the other.
C. Enzyme Efficiency (s–1(mol.l–1) –1)
The ratio of kcat/Km is defined as the catalytic efficiency or a measure of substrate
specificity. When thekcat is markedly greater than k–1, it indicates that the catalytic
process is extremely fast sequel to the efficiency of the enzyme to bind the substrate.
Based on the laws of diffusion, the upper limit forsuch rates, as determined by the
frequency of collisions between the substrate andthe enzyme, is between 108and 109.
Some enzymes are very efficient with their catalytic rate approaching this range, e.g.,
fumarase,2.3 × 108s–1(mol.l–1) –1
D. vmax
vmaxis the maximum velocity that an enzyme could achieve. Measurement of vmaxis
theoretical because at a given time, it would require all enzyme molecules to be
tightly bound to their substrates. vmax is approached at high [A] but never reached as
can be seen in Figure 5.

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 Figure 5: Change in velocity with concentration of substrate

DETERMINATION OF MICHAELIS–MENTEN PARAMETERS

Determination of M-M kinetic parameters is usually done graphically.

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