KINETICS OF ENZYME-CATALYZED REACTIONS

Introduction

Enzymes are biological catalysts that are protein molecules in nature and absolutely

essential in biochemical reactions. Like every other catalyst they increase the rate of reaction

without themselves undergoing permanent chemical changes.

The catalytic ability of enzymes is due to its particular protein structure. A specific

chemical reaction is catalyzed at a small portion of the surface of an enzyme, which is known as

active site. Enzyme reactions are different from chemical reactions, as follows:

1. An enzyme catalyst is highly specific, and catalyzes only one or a small number of

chemical reactions. A great variety of enzymes exist, which can catalyze a very wide

range of reactions.

2. The rate of an enzyme-catalyzed reaction is usually much faster than that of the same

reaction when directed by non-biological catalysts. Only a small amount of enzyme is

required to produce a desired effect.

3. The reaction conditions (temperature, pressure, pH, and so on) for the enzyme reactions

are very mild.

4. Enzymes are comparatively sensitive or unstable molecules and require care in their use.

Enzymes are named with the suffix ‘-ase’ to the name of the substrate with which the enzyme

functions e.g. Lactase, Maltase.

The six classes of enzymes are defined on the basis of the types of reactions they

catalyze.
 1. Oxidoreductase

 Catalyzes redox reactions

 Acts on CH – OH group of substrates

 Specific substrate is ethyl alcohol

2. Transferase

 Catalyzes transfer of functional groups

3. Hydrolase

 Splits chemical bonds by addition of water (hydrolysis)

4. Lyase

 Splits chemical bonds without using water (not a hydrolysis reaction).

5. Isomerase

 Rearranges atoms within a molecule.

6. Ligase

 Forms a chemical bond between two atoms.

ENZYME KINETICS

Enzyme kinetics deals with the rate of enzyme reaction and how it is affected by various

chemical and physical conditions. Kinetic studies of enzymatic reactions provide information

about the basic mechanism of the enzyme reaction and other parameters that characterize the
properties of the enzyme. The rate equations developed from the kinetic studies can be applied in

calculating reaction time, yields, and optimum economic condition, which are important in

the design of an effective bioreactor.

Assume that a substrate (S) is converted to a product (P) with the help of an enzyme (E)

in a reactor as

If you measure the concentrations of substrate and product with respect to time, the product

concentration will increase and reach a maximum value, whereas the substrate concentration will

decrease as shown in figure below

The rate of reaction can be expressed in terms of either the change of the substrate Cs or the

product concentrations CP as follows:

 In order to understand the effectiveness and characteristics of an enzyme reaction, it is

important to know how the reaction rate is influenced by reaction conditions such as

substrate, product, and enzyme concentrations. If we measure the initial reaction rate at

different levels of substrate and enzyme concentrations, we obtain a series of curves like the one

shown below.

From these curves we can conclude the following:

1. The reaction rate is proportional to the substrate concentration (that is, first-order reaction)

when the substrate concentration is in the low range.

2. The reaction rate does not depend on the substrate concentration when the substrate

concentration is high, since the reaction rate changes gradually from first order to zero order as

the substrate concentration is increased.

3. The maximum reaction rate rmax is proportional to the enzyme concentration within the range

of the enzyme tested.

Henri observed this behavior in 1902 (Bailey and Ollis, p. 100, 1986) and proposed the

rate equation

where rmax and KM are kinetic parameters which need to be experimentally determined. Eq. (2.4)

expresses the three preceding observations fairly well. The rate is proportional to CS (first order)

for low values of CS, but with higher values of Cs, the rate becomes constant (zero order) and

equal to rmax. Since Eq. (2.4) describes the experimental results well, we need to find the kinetic

mechanisms which support this equation.

Brown (1902) proposed that an enzyme forms a complex with its substrate. The complex

then breaks down to the products and regenerates the free enzyme. The mechanism of one

substrate-enzyme reaction can be expressed as

 The reaction rate equation can be derived from the preceding mechanism based on the

following assumptions:

1. The total enzyme concentration stays constant during the

reaction, that is, CE0= CES+ CE

2. The amount of an enzyme is very small compared to the amount of substrate. Therefore,

the formation of the enzyme substrate complex does not significantly deplete the

substrate.

3. The product concentration is so low that product inhibition may be considered negligible.

In addition to the preceding assumptions, there are three different approaches to derive

the rate equation:

1. Michaelis-Menten approach (Michaelis and Menten, 1913): It is assumed that the

product-releasing step, Eq. (2.6), is much slower than the reversible reaction, Eq. (2.5),

and the slow step determines the rate, while the other is at equilibrium. This is an

assumption which is often employed in heterogeneous catalytic reactions in chemical

kinetics.

2. Briggs-Haldane approach (Briggs and Haldane 1925): The change of the intermediate

concentration with respect to time is assumed to be negligible, that is, d(CES)/dt = O. This

is also known as the pseudo-steady-state (or quasi-steady-state) assumption in chemical

kinetics and is often used in developing rate expressions in homogeneous catalytic

reactions.

3. Numerical Solution
  Michaelis-Menten Approach

If the slower reaction determines the overall rate of reaction, the rate of product formation

and substrate consumption is proportional to the concentration of the enzyme-substrate complex

as:

dC p −dC s
r= = =k 3 C ES
dt dt

From the assumption that the first reversible reaction is in equilibrium. Then, the forward

reaction is equal to the reverse reaction so that:

k 1 C s C E=k 2 C ES

If we assume that the total enzyme contents are conserved, the free-enzyme concentration CE

can be related to the initial enzyme concentration CE0

C E 0 =C E +C ES

By substituting for CE and rearranging for CES, we obtain:

CE 0CS
C ES =
k2
+Cs
k1

Substitution results in the final rate equation

 dC p −dC s k 3 C E 0 CS
r= = =
dt dt k2
+C s
k1

r m ax C s
r=
k M +C s

Which is known as Michaelis-Menten equation.

k2 CsCE
k M= =
k 1 C ES

r max =k 3 C E 0

 Briggs-Haldane Approach

The rates of product formation and of substrate consumption are:

dC p
=k 3 C ES
dt

−dC s
=k 1 C s C E −k 2 C ES
dt

The change of the intermediate concentration with respect to time is assumed to be

negligible, that is, d(CES)/dt = O.

dC ES
=k 1 C s C E−k 2 C ES −k 3 C ES=0
dt

Again
 dC p −dC s
r= = =k 3 C ES
dt dt

C E 0 =C E +C ES

Substituting and rearranging

C E0CS
C ES =
k 2 +k 3
+C s
k1

Results in

dC p −dC s k 3 C E 0 C S
r= = =
dt dt k 2+ k 3
+C s
k1

r max C s
¿
k M +C s

which is the same as the Michaelis-Menten equation, except that the meaning of KM is

different. In the Michaelis-Menten approach, is equal to the dissociation constant

k2/k1while in the Briggs-Haldane approach, it is equal to (k2+k3)/k1.This can be simplified

to k2/k1if k2» k3which means that the product releasing step is much slower than the

enzyme-substrate complex dissociation step. This is true with many enzyme reactions.

Since the formation of the complex involves only weak interactions, it is likely that the

rate of dissociation of the complex will be rapid. The breakdown of the complex to yield
 products will involve the making and breaking of chemical bonds, which is much slower

than the enzyme-substrate complex dissociation step.

Evaluation of Kinetic Parameters

In order to estimate the values of the kinetic parameters, we need to make a series of

batch runs with different levels of substrate concentration. Then the initial reaction rate can be

calculated as a function of initial substrate concentrations. The results can be plotted graphically

so that the validity of the kinetic model can be tested and the values of the kinetic parameters can

be estimated.

The Michaelis-Menten equation is usually rearranged so that the results can be plotted as

a straight line. Some of the better-known methods are presented here. The Michaelis-Menten

equation, can be rearranged to be expressed in linear form. This can be achieved in three ways:

Cs KM Cs
= +
r r max r max

An equation of the form of above was given by Langmuir (Carberry, 1976) for the treatment of

data from the adsorption of gas on a solid surface. If the Michaelis-Menten equation is

applicable, the Langmuir plot will result in a straight line, and the slope will be equal to

1/rmax.The intercept will be Km/rmaxas shown in below.

 1 1 KM 1
= +
r r max r max C s

Similarly, the plot of 1/rversus 1/Cswill result in a straight line according to equation above and

the slope will be equal to KM/rmax,the intercept will be 1/ rmax as shown in Figure below. This plot

is known as Lineweaver-Burk plot (Lineweaver and Burk, 1934).

r
r =r max −K M
Cs
The plot of r versus r/Cs will result in a straight line with a slope of −K M and an intercept of rmax

as shown in Figure below. This plot is known as the Eadie-Hofsteeplot (Eadie, 1942; Hofstee,

1952).

The Lineweaver-Burk plot is more often employed than the other two plots because it

shows the relationship between the independent variable Cs and the dependent variable r.

Enzyme Inhibition

A modulator (or effector) is a substance which can combine with enzymes to alter their

catalytic activities. An inhibitor is a modulator which decreases enzyme activity. It can decrease

the rate of reaction either competitively, noncompetitively, or partially competitively.

 Competitive Inhibition

Since a competitive inhibitor has a strong structural resemblance to the substrate,

both the inhibitor and substrate compete for the active site of an enzyme. The formation of an
enzyme-inhibitor complex reduces the amount of enzyme available for interaction with the

substrate and, as a result, the rate of reaction decreases. A competitive inhibitor normally

combines reversibly with enzyme. Therefore, the effect of the inhibitor can be minimized by

increasing the substrate concentration, unless the substrate concentration is greater than the

concentration at which the substrate itself inhibits the reaction. The mechanism of competitive

inhibition can be expressed as follows:

If the slower reaction, the product formation step, determines the rate of reaction according to the

Michaelis-Menten assumption, the rate can be expressed as:

r p =k 5 C ES

The enzyme balance gives

C E 0 =C E +C ES +C EI

From the two equilibrium reactions,

C E CS k 2
= =K S
C ES k1

C E C I k4
= =K I
C EI k3
Where KSand KI are dissociation constants. Combining the preceding four equation to eliminate

CE, CES, and CEI yields

r max C s
r p=
C S −K MI

Where

CI
K MI =K S (1+ )
KI

 Non- competitive Inhibition

Noncompetitive inhibitors interact with enzymes in many different ways. They can bind to

the enzymes reversibly or irreversibly at the active site or at some other region. In any

case the resultant complex is inactive. The mechanism of noncompetitive inhibition can be

expressed as follows:
 Since substrate and inhibitor do not compete for a same site for the formation of enzyme-

substrate or enzyme-inhibitor complex, we can assume that the dissociation constant for the

first equilibrium reaction is the same as that of the third equilibrium reaction, as

As shown in the previous section, the rate equation can be derived by employing the

Michaelis-Menten approach as follows: where

r I , max Cs
r p=
C S−K S

Where

r max
r I ,max =
1+ C I / K I

Therefore, the maximum reaction rate will be decreased by the presence of a noncompetitive

inhibitor, while the Michaelis constant Ks will not be affected by the inhibitor. The graphical

consequences of noncompetitive inhibition are shown below. Note that making these plots

enables us to distinguish between competitive and noncompetitive inhibition.

 Several variations of the mechanism for noncompetitive inhibition are possible. One case is

when the enzyme-inhibitor-substrate complex can be decomposed to produce a product and the

enzyme inhibitor complex. This mechanism can be described by adding the following slow

reaction

This case is known as partially competitive inhibition.

Other influences

The rate of an enzyme reaction is influenced by various chemical and physical

conditions. Some of the important factors are the concentration of various components (substrate,

product, enzyme, cofactor, and so on), pH, temperature and shear.

Effect of pH

The rate of an enzyme reaction is strongly influenced by the pH of the reaction solution.

The typical relationship between the reaction velocity and pH shows a bell-shaped curve. The

optimum pH is different for each enzyme.The reason that the rate of enzyme reaction is

influenced by pH can be explained as follows:

1. Enzyme is a protein which consists of amino acid residues (that is, amino acids minus

water).

2. The amino acid residues possess basic, neutral, or acid side groups which can be

positively or negatively charged at any given pH.

3. An enzyme is catalytically active when the amino acid residues at the active site each

possesses a particular charge. Therefore, the fraction of the catalytically active enzyme

depends on the pH.

Effect of temperature

The rate of a chemical reaction depends on the temperature according to

Arrhenius equation as

k = A0 e−E / RT

Consequently, when Ink is plotted versus 1/T, a straight line with slope –E/R is obtained.

The temperature dependence of many enzyme-catalyzed reactions can be described by

the Arrhenius equation. An increase in the temperature increases the rate of reaction, since the

atoms in the enzyme molecule have' greater energies and a greater tendency to move. However,
the temperature is limited to the usual biological range. As the temperature rises, denaturation

processes progressively destroy the activity of enzyme molecules. This is due to the unfolding of

the protein chain after' the breakage of weak (for example, hydrogen) bonds, so that the overall

reaction velocity drops. For many proteins, denaturation begins to occur at 45 to 50°C. Some

enzymes are very resistant to denaturation by high temperature, especially the enzymes isolated

from thermophilic organisms found in certain hot environments.

Effect of Shear

Enzymes had been believed to be susceptible to mechanical force, which disturbs the

elaborate shape of an enzyme molecule to such a degree that denaturation occurs. The

mechanical force that an enzyme solution normally encounters is fluid shear, generated either by

flowing fluid, the shaking of a vessel, or stirring with an agitator. The effect of shear on the

stability of an enzyme is important for the consideration of enzyme reactor design, because the

contents of the reactor need to be agitated or shook in order to minimize mass transfer

resistance.

(Dutta, 2008) (Fundamentals of Biochemical engineering, Rajiv Dutta 2008)