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ENZYME KINETICS LECTURE NOTES Second Edition

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ENZYME KINETICS LECTURE
NOTES
Second Edition

Andreas Kukol
 Copyright © 2015, 2017 Andreas Kukol

Create Space Independent Publishing, Charleston, USA

All rights reserved

ISBN-13: 978-1548471019
ISBN-10: 1548471011
Preface
The ‘Lecture Notes’ cover the topic of enzyme kinetics for a three-year undergraduate
programme in bioscience. Many parts are relevant for all bioscience degree courses, such as
pharmacology or biomedical sciences, while some of the advanced areas are more suitable
for final year biochemistry, molecular biology or biotechnology courses.

The text does not assume much background knowledge except familiarity with the concepts
of molar concentrations, chemical reactions and some basic mathematical concepts (the four
basic arithmetic operations, fractions, exponents and logarithms, the notion of solving an
equation). All other background maths and other concepts are explained in the text.

Various sections in the second edition have been expanded with textboxes providing
additional explanations of spectrophotometry, calculations with the Arrhenius equation,
how to derive rate equations for complex mechanisms, Cleland diagrams and matrices,
eigenvalues and –vectors. Two new sections were added about enzyme inhibitor constants
in pharmacology and allosteric enzymes.
Contents
1 Introduction..............................................................................................................................................1

1.1 Enzyme assays ..................................................................................................................................2

1.2 Application of enzyme assays .......................................................................................................2

1.3 Practical aspects................................................................................................................................4

1.3.1 Pre-steady state kinetic measurements................................................................................8

2 Chemical Kinetics .................................................................................................................................. 11

2.1 Definition of the reaction rate...................................................................................................... 12

2.2 Rate law and reaction mechanism..............................................................................................13

2.2.1 Order of rate laws ................................................................................................................... 13

2.2.2 Relationship between rate laws and reaction mechanisms ........................................... 14

2.3 Integrating the rate law ................................................................................................................ 16

2.4 Rate laws for complex reaction mechanisms ........................................................................... 20

2.5 Compound rate laws .....................................................................................................................22

2.6 The rate limiting step .................................................................................................................... 22

2.7 Temperature dependence of reaction rates .............................................................................. 24

2.8 A molecular picture of chemical reactions ............................................................................... 27

3 Enzyme Kinetics .................................................................................................................................... 29

3.1 The transition state theory applied to enzymes ...................................................................... 29

3.2 The reaction mechanism of alkaline phosphatase .................................................................. 30

3.3 The Michalis-Menten equation ................................................................................................... 33

3.3.1 Interpretation of the Michaelis-Menten equation............................................................37

3.3.2 Enzyme activity and enzyme units.....................................................................................39

3.4 Analysis of enzyme kinetic data ................................................................................................. 40

3.4.1 Obtain initial reaction rates .................................................................................................. 41

3.4.2 The hyperbolic plot ................................................................................................................ 42

3.4.3 The Lineweaver-Burk plot .................................................................................................... 43

 3.4.4 Non-linear curve fitting......................................................................................................... 46

3.4.5 Fitting of differential equations ...........................................................................................47

3.5 Reversible inhibition .....................................................................................................................47

3.5.1 Competitive inhibition .......................................................................................................... 48

3.5.2 Uncompetitive inhibition ...................................................................................................... 49

3.5.3 Mixed and non-competitive inhibition .............................................................................. 51

3.5.4 Data analysis for reversible inhibition ............................................................................... 52

3.5.5 Inhibitor binding sites for reversible inhibition ...............................................................54

3.5.6 Inhibitor constants in pharmacology ................................................................................. 55

3.6 Deviations from Michaelis-Menten kinetics ............................................................................ 58

3.6.1 Substrate inhibition ................................................................................................................ 58

3.6.2 Product inhibition................................................................................................................... 59

3.6.3 Sigmoid kinetics and cooperativity .................................................................................... 60

3.7 Enzyme reactions with two substrates ......................................................................................62

3.7.1 Ternary complex mechanisms .............................................................................................62

3.7.2 Double displacement/ping-pong reaction......................................................................... 67

3.8 Allosteric enzymes.........................................................................................................................70

4 Single-Molecule Kinetics .....................................................................................................................75

4.1 Single molecule chemical kinetics ..............................................................................................75

4.1.1 Systems of unimolecular reactions .....................................................................................76

4.1.2 Bimolecular reactions ............................................................................................................ 82

4.2 Single molecule enzyme kinetics ................................................................................................83

4.2.1 The single molecule Michaelis-Menten equation ............................................................85

4.3 Stochastic simulations................................................................................................................... 87

 1 Introduction

1 INTRODUCTION
Enzyme kinetics is concerned with the influence of enzymes on chemical reaction rates.
Enzymes are biological catalysts that speed up chemical reactions. They occur naturally in
biological systems, although in modern bio-technology they are also used in isolation to
facilitate reactions of organic chemistry or to participate in the detection of analytes.

Kinetics of chemical reactions is a topic of physical chemistry concerned with chemical

reaction rates, thus enzyme kinetics in particular belongs to the area of biophysical
chemistry.

Enzymes are to a large extent protein molecules, although some enzymes are made from
RNA and are referred to as ribozymes. An example of the three-dimensional structure of an
enzyme is shown in Figure 1. Enzymes are nanometre sized particles that usually have a
surface-accessible cleft or groove that forms the active site.

Figure 1: Three-dimensional structure of the enzyme alkaline phosphatase (PDB-ID: 1ALK). The active site is
highlighted in green. The approximate diameter of this globular protein is 6.8 nm (nanometre).

Proteins, which form the majority of enzymes, are polymers of amino acids. Twenty
different proteinogenous amino acids exist as the building blocks of proteins; amino acids
are joined by the peptide bond into a long chain (449 amino acid residues in Figure 1) that is

1
 Chemical Kinetics

1
[B] in concentr.

0.8
0.6
units

0.4
0.2
0
0 10 20 30 40

1 Reaction 2
reaction rate

0.8
Individual

Reaction 1 (forward)
0.6
0.4 Reaction 1 (backward)
0.2
0
0 10 20 30 40
time/s
Figure 11: Results of a numerical integration. Top: the concentration of the final product B
plotted against time. Bottom: the individual reaction rate for each reaction.

Figure 11 shows that reaction two is always slower than the other reactions. Furthermore,
after approximately 2.5 s the forward and backward reaction rates of reaction one are equal.
Equal forward and backward reaction rates mean that an equilibrium has established itself.
The particular reaction introduced above is an example of a pre-equilibrium. The
approximation of a fast pre-equilibrium is sometimes used to simplify the kinetic analysis of
complex reaction schemes. In section 3.3 the steady-state approximation will be introduced
as another way of simplifying the kinetic analysis.

2.7 Temperature dependence of reaction rates

The reaction rate increases with increasing temperature; as a rule of thumb raising the
temperature by 10 K doubles the reaction rate. The temperature dependence of a rate
constant is described by the Arrhenius equation:

24
 Chemical Kinetics

 E 
k  A exp  A 
 RT 

with k: rate constant, A: pre-exponential factor (in the units of the rate constant), EA:
activation energy (in J mol-1), R = 8.314 J K-1 mol-1 and T: temperature in Kelvin.

The pre-exponential factor A is related to the collision between molecules and to internal
motions (in a bimolecular reaction) or to internal motions of the molecule only (in a
unimolecular reaction). While there are theories that allow calculation of A from molecular
properties, it is usually determined through experiments. When the ratio of two rate
constants at two different temperatures T1 and T2 is considered, A cancels out (see Box 5 ):

k2 E  1 1 
 exp  a   
k1 R  T1 T2 

The activation energy can be interpreted as an energy barrier that must be overcome before
the reaction proceeds to products. The point at the top of this energy barrier is called the
transition state. The diagram in Figure 12 illustrates the concept of activation energy.

Free
enthalpy Ea
G/J A

ΔG
B

Reaction progress

Figure 12: A reaction energy profile for the exergonic reaction A  B. Ea is the activation energy and ΔG is the
free enthalpy change of the reaction.

As a reaction proceeds the reactants must overcome this energy barrier. With increasing
temperature the thermal energy of the reactants increases, which makes it more likely for
them to overcome the energy barrier. This temperature dependence is described by the
Arrhenius equation.

25
 Chemical Kinetics

The height of the activation barrier is related to the rate constant as

EA = (ln A/k)/RT, i.e. the lower the rate constant, the higher the activation barrier. Some
textbooks state incorrectly that for multistep reactions the rate limiting step is always the
reaction with the highest activation barrier (see section 2.6).

Box 5: The Arrhenius equation expressed as the ratio of two rate constants.

The ratio of rate constants k1 at temperature T1 and k2 at temperature T2 can be written

as:

 E 
A exp  A 
k2
  RT2  (the pre-exponential factor A cancels out)
k1  E 
A exp  A 
 RT1 

 E 
exp  A 
  RT2  (the exponential is resolved exp(A)/exp(B) = exp(A-B) )
 E 
exp  A 
 RT1 
 E  E 
 exp  A    A   (rearrange)
 RT2  RT1  
E E 
 exp  A  A  (extract common factor EA/R)
 RT1 RT2 

 E  1 1 
 exp A    
 R  T1 T2  

26
 3 Enzyme Kinetics

3.2 The reaction mechanism of alkaline phosphatase

Alkaline phosphatase is an enzyme that hydrolyses esters of phosphoric acid into the
corresponding alcohol and phosphoric acid. The kinetics of enzymes is often studied with
model substrates (see chapter 1.2) that yield a colour, so the progress of the reaction can be
followed with a spectrophotometer. In case of alkaline phosphatase, the substrate p-nitro-
phenylphosphate is often used that is hydrolysed into p-nitro-phenol and hydroxy-
phosphate:

- O Alkaline
- O phosphatase
O P - -
+ O O - O
N O O
+
O
+ H2O N OH
+ P
O
-
O HO

R-O-P R-OH Pi

The mechanism of this reaction is illustrated in the following figures (reviewed in Holtz &
Kantrowitz, 1999) :
NH 2 R
2+ H Arg166
Zn 2+
+ Zn O
O H 2N NH - H2 N
H O
P - NH Arg166
O +
O H2N
O Ser102
2+
Zn -
H O Ser102
2+
- H Zn
O H
H
O - O
2+
O Mg
O
CH 3 - 2+
O Mg
HO
Asp51 CH 3
-
O HO
Asp51
Thr155 -
O
O
Thr155
Glu322 O

Glu322

E + R-O-P E ∙ R-O-P

Figure 15A: Initially the active site of the enzyme contains one water molecule and hydroxyl-ion (OH-) as well
as the two Zn2+ and one Mg2+ ion as co-factors. Hydroxyl ions are formed under alkaline conditions, which
explains why the enzyme prefers an alkaline pH of >10. In the first step the negatively charged substrate
replaces the water molecule and associates with the enzyme facilitated by interactions with the positively
charged Arg166 and the two Zn2+ ions. This illustrates the formation of the enzyme-substrate complex. The
hydroxyl-ion extracts a proton from the residue Ser102.

30
 3 Enzyme Kinetics

3.3.1 Interpretation of the Michaelis-Menten equation

The Michaelis-Menten equation provides the dependence of the initial reaction rate v0 on
substrate concentration. The plot of v0 against substrate concentration is called a hyperbolic
plot or saturation plot and shown in Figure 17.

Figure 17: A plot of the initial reaction rate v0 against substrate concentration according to the Michaelis-
Menten equation with v max = 1.0 concentration units/time. The substrate concentration is shown in multiples of
KM, so that KM is equal to 1.0 in this example. Normally substrate concentrations would be in the units of μM or
mM and v0 in the units of μM/s or mM/s.

The typical feature of the saturation plot is the initial steep rise of v0 with increasing
substrate concentrations followed by a gradual flattening of the curve. The maximum initial
reaction rate vma x is approached asymptotically. In the limit of infinite substrate
concentration the vma x of 1.0 concentration/time is reached. Note that for the example shown
in Figure 17 even at substrate concentrations of 8 KM the maximum initial reaction rate has
not been reached.

The aim of enzyme kinetic data analysis is to determine KM and vma x. If we know the
concentration of active enzyme sites, we can use vmax to calculate the turnover number that is
the number of reaction the enzyme performs per unit time. This is also known as kc at:

kka t = vma x / [E]T

Note that for multimeric enzymes, the concentration of active sites [E]T is a multiple of the
enzyme concentration, while for enzymes with one active site [E]T is equal to the
concentration of active enzyme. Furthermore, the concentration of active enzyme is often
not equal to the total protein concentration, as proteins are sensitive biological materials that

37
 3 Enzyme Kinetics

3.4.1 Obtain initial reaction rates

The primary data is usually a set of product or substrate concentrations obtained at different
time points as shown in Figure 18.

A B
concentration

concentration
c

c t
t
time time

Figure 18: Change in product concentration over time as measured in an enzyme assay. The initial reaction rate
is taken from the gradient of the linear phase as Δc/Δt. A) Example of a slow (compared to the timescale of the
measurement) enzyme with a long linear phase B) Example of a fast enzyme with a short linear phase.

From the linear phase the initial reaction rate is determined as v0 = Δc/Δt. If the enzyme is
fast compared to the time scale of the measurement method as in Figure 18B, the
determination of the initial rate becomes more inaccurate. Since at higher substrate
concentration the reaction rate increases the time scale of the measurement may need to be
changed (measuring at shorter time intervals).

For automated analysers used in biomedical laboratories often a two-point estimate of the
reaction rate is taken. In the development of enzyme assays for automated analysers
conditions need to be found that produce a linear concentration increase (or decrease) for a
sufficient amount of time. The conditions that may be changed could be pH (different than
optimal pH), temperature or a different substrate that gives lower reaction rates.

41
 3 Enzyme Kinetics

Figure 24: The hyperbolic plot of the initial reaction rate v0 against substrate concentration [S]
for an enzyme with vmax = 1.0 concentration/time and KM = 1.0 concentration units under the
influence of various types of reversible inhibitors.

A good diagnostic tool is the shape of the Lineweaver-Burk plots as shown in the sections
above. At the same time the apparent kinetic constants should be determined and based on
the influence of increasing inhibitor concentrations the mechanism of inhibition may be
determined (Table 3).

Table 3: The change of the apparent enzyme kinetic constants with increasing inhibitor concentrations for
different mechanisms of inhibition (‘+’ denotes increase, ‘−‘ denotes decrease and ‘0’ denotes no change).

Mechanism KM’ vmax’

Competitive + 0

un-competitive − −

Mixed +/- −

non-competitive 0 −

53
 3 Enzyme Kinetics

Figure 26: An example of the determination of an IC50 value for an inhibitor. The % of enzyme activity is
plotted against the logarithm of the inhibitor concentration. The IC50 value can be determined graphically or
better through non-linear curve fitting.

If the mechanism of inhibition, KM of the enzyme, KI of the inhibitor and the substrate
concentration is known, the IC50 value can be calculated with the Cheng-Prusoff
relationships, or alternatively KI can be calculated from IC50 by rearringing the equations:

 [S ] 
Competitive inhibition: IC50  K I 1  
 KM 
 K 
Uncompetitive inhibition: IC50  K I 1  M 
 [S} 
[S ]  K M
Mixed inhibition: IC50 
[S ] K M

K I' KI
Non-competitive inhibition: IC50  K I

56
 3 Enzyme Kinetics

Box 9: An alternative display of two-substrate mechanisms using Cleland diagrams.

Compulsary order:

A B B’ A’

E E
EA EAB EA’B’ EA’

Random order:

A B B’ A’

EA EAB EA’B’ EA’

E E
EB EB’

B A A’ B’

Ping-pong (compulsary order):

AX A B BX

E E
E·AX EX EX·B E·BX

69
 3 Enzyme Kinetics

3.8 Allosteric enzymes

Allosteric enzymes have a binding site for an effector molecule that is spatially distant from
the active site. This effector may be an activator or inhibitor. The binding site is called
allosteric site; the effector is called allosteric effector. The allosteric effector is called heterotropic,
if it is a different molecule than the substrate and homotropic, if it is the substrate. Indeed, the
substrate can be an allosteric effector, if there is a binding site for the substrate that is
spatially different from the active site of the enzyme.

An example of an allosteric enzyme is aspartate transcarbamoylase (ATC) that catalyses the

first step in the synthesis of pyrimidine nucleotides. ACT is a multimeric protein that has
twelve subunits; the catalytically active unit is a trimer C3, and the regulatory unit is a dimer
R2, so the overal subunit composition is 2 C3 + 3 R2 = C6R6. Each catalytic trimer unit has three
active sites and the regulatory dimer unit has two allosteric sites. The allosteric sites can
bind the nucleotide ATP, an allosteric activator, and the nucleotide CTP, an allosteric
inhibitor.

Figure 33: Structure of aspartate transcarbamoylase (PDB-ID: 4FYW). The two catalytic trimers are shown in
green with active sites highlighted in gold. The three regulatory dimers are shown in grey with bound CTP in
blue. B shows the structure at a different angle, rotated 90° to the front.

From the structure shown in Figure 33 it is clear that the regulatory sites (blue) are spatially
distinct from the active sites (gold), hence ATC is an allosteric enzyme. Most allosteric en-
zymes (but not all) show cooperativity of substrate binding that can be identified from
sigmoid initial rate kinetics (see page 60). An allosteric activator such as ATP shifts the
70
 4 Single-Molecule Kinetics

4.3 Stochastic simulations

While numerical integration of differential equations in ensemble kinetics is sometimes

referred to erroneously as simulation, the stochastic behaviour of single molecules cannot be
obtained from numerical integration. Stochastic simulations take into account each molecule
in the system and the various reactions (or transitions) available to each molecule. The
original stochastic simulation algorithm for chemical kinetics was suggested by Gillespie
(1977). Consider a molecule A that can undergo various reactions (described by rate
constants k1 , k2, .., ki):

k2
k1
ki
A

The Gillespie algorithm entails drawing a random number that is exponentially distributed
with the decay constant ∑ki ; this specifies the time the molecule stays in state A, which is on
average <t> = 1/∑ki. Another random number determines, which reaction takes place, while
the probability for each reaction r is given as kr/∑ki. The algorithm is summarised in Box 11.
The original algorithm by Gillespie, often called the direct method, has been modified to
increase the speed of the simulation as well as approximate methods with further
performance increase, such as tau-leaping methods, have been developed (Gillespie, 2007).

Box 11: The Gillespie algorithm for stochastic simulations.

Initialise (set initial numbers)

Draw random variable t (exponentially distributed with <t> = 1/∑ki)*

Draw another random integer variable to determine which reaction occurs
(the probability for each reaction r is: kr/∑ki )*
Update molecule numbers
Increment time T = T + t
Until specified end-time is reached.

*) Note that in case of second order reactions the instantaneous number of molecules
of one of the reaction partners would be taken into account, e.g. ki nA nB for the
reaction A + B  …

87

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