UNIT 18 ENZYME KINETICS OF SINGLE SUBSTRATE REACTIONS

18.1 PREAMBLE

Enzyme kinetics presents a similar approach to adsorption reaction where there is

an intermediate step before the final formation of the products. It can be
understood that enzymes are weak catalysts that can easily be disturbed by the
conditions of the reaction. The temperature, pH and other factors tend to disturb
the reaction by way of slowing it down or accelerating it. We see that some
enzymes are not comfortable with acidic conditions while other not comfortable
with basic conditions. Most of them prefer neutral conditions of pH and mild
conditions of temperature as too much heat denatures the enzymes. This unit
examines reactions at all conditions.

18.2 OBJECTIVES

1. Present enzymatic reactions

2. Develop some approaches of solving problems with enzymes
3. Evaluate the effect of temperature and pH on the enzyme activity

18.3 LEARNING OUTCOMES

1. Develop the understanding of how the enzymes promote reactions

2. Apply the approaches in solving enzyme kinetic problems
3. Examine the effects of temperature and pH on the enzymatic reactions

18.4 DEFINITIONS

Enzyme: Biochemical catalyst that speeds up the chemical reaction

Substarate: The food that microorganisms use to increase their activity

Michaelis constant,Km: This is a constant that is used to understand how

biochemical reactions behave

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Maximum reaction rate, Vmax: This the maximum reaction rate for the
biochemical reaction

18.5 ENZYME KINETICS

Enzymes act like and are catalysts that promote the biochemical reactions. Like in inorganic
systems enzymes remain unchanged after the chemical reactions. However they also get
affected by a number of factors; pH, temperature and impurities which can inhibit the activity
of the enzymes. At high temperatures catalysts can be destroyed by heat, while extreme pH
values can do the same. The impurities can react with the enzyme thereby disturbing the
activity and reducing the potency of the catalyst.

Some enzymes may include the ones as listed in Table 1 as follows;

No Enzyme Reaction Temperature (oC)

1 Bromelain 0 - 37
2 Fumarase 0 - 37
3 Ribonuclease 0 - 37
4 Trypsin 0 - 37

18.5.1 Properties of enzymes

The property is a characteristic of the enzyme that makes it to stand out as it functions during
the biochemical reaction. The property does not change over the time. Here are some of the
properties of the enzymes:

1. Efficiency is higher at room temperature

2. Specificity: They are specific in action e.g specific for one type of molecule e.g glucose
oxidase oxidises D(+) glucose only
3. Versatility: Versatility of enzymes depends on the type of reaction
a. e.g.Hydrolases for hydrolysis reactions
b. e.g. Lyases for remival of groups from substrates
c. e.g Isomerases for isomerisation reaction
d. e.g. Oxidoreductase for oxidation-reduction reactions
4. They can be controlled by environmental conditions
5. They are relatively unstable as the functional tertiary structure is held by weak forces

18.5.2 Factors Responsible for Enzyme Kinetics

1. Some of the factors that responsible for enzyme kinetics are as follows:

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 2. Enhance the rate of reaction but do not alter thermodynamic equilibrium
3. Molecules must collide for the reaction to take place
4. The collision must be in the correct orientation
5. There must be sufficient activation energy
6. There must always be enzyme-substrate and enzyme-product-substrate or complex

The first step is he formation of the enzyme-substrate complex (ES). This is more stable than
enzyme alone as shown in (1)

E  S  ES 
 E  P (1)

E=Enzyme

S=Substarte

P=Product

This is known as the Briggs Haldane equation.

The decomposition of ES to E+P is not reversible

The decomposition from ES to E+S is reversible

Therefore the decomposition of ES is rate limiting

It is also assumed that decompositions are at steady state

The concentration of ES is constant

d ( ES )
Its rate of change is equal to zero. Therefore 0
dt

Figure 2 below shows the enzyme catalysed reaction as it proceeds during the process.

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Figure 1 Concentration verses steady state time

1. S decreases during biochemical reaction

2. P increases during the reaction
3. E decreases and remains steady and increases after steady state
4. ES increases and remains steady and decreases after steady state

As enzyme substrate is formed by one pathway it disappears by two of formation and product
formation as given in (2) below

d ( ES )
k f 1ES  k f 2 ES  kr1ES  0
dt

Kf1 is the rate constant for ES while kf2 is the rate for decomposition of ES forward reaction. The
kr1 is the rate constant for the reverse reaction first step

Since ES  Etot  E (3)

Substitute (2) in (3) we get the following

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 Etot S
ES  (4)
k  kf2
S  r1
kf1

If R0 is the initial rate of reaction which is given by

R0  k f 2 ES (5)

Substitute (4) in (5) we get the following

k f 2 Etot S
R0  (6)
k kf 2
S  r1
kf1

Introduce Michaelis Constant Km in (6) we get the following

k f 2 Etot S
R0  (7)
S  Km

k r1  k f 2
Km  (8)
kf1

When we introduce the term Vmax (the maximum rate of reaction) given by the following

Vmax  k f 2 Etot (9)

We find that the value of R0 becomes as follows

Vmax S
R0  (10)
S  Km

A plot of R verses S gives the figure below

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Figure 2 Vmax verses substrate (S)
kcat is the rate constant for the decomposition of S

Figure 2 above shows the reaction rate versus substrate concentration

It is first order at low concentration of substrate

It is zero order at high concentration of substrate

Kf2 = kcat in simple situations but in complex situations kcat will be the sum of decomposition
terms.

Km is the concentration required to give half the value of maximum rate

The values of Km range from 0.01 to 20x10-3 M or 10-5 to 0.02 kmol/m3

18.5.3 Kinetics of Two Substrate Reactions

Most enzymes catalyse two interacting substrates

Such reactions show complicated kinetics than single substrate kinetics

Enzymes catalyzing two substrate reactions include the following:

1. Transferase reactions where transfer of functional groups from one substrate to another
takes place e.g. amino-transferase and phosphorylases
2. Dehydrogenases reactions where NAD(P) and NAD(P)H are co-substrates with carbon
substrates e.g alcohol dehydrogenases (ADH)

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 3. NAD means Nicotinamide Adenosine Dinucleotide which acts as a coenzyme or
cosubstrate
L-Lactate: NAD+ oxidoreductase
D-Lactate: NAD+ oxidoreductatese
Ethanol: NAD+ oxidoreductase

CH 3CH 2OH  NAD CH 3CHO  NADH  H 

ADH
(11)

Kinetics of two substrate reactions is more complex due to increased number of possible
enzyme substrate complexes

For example in the following reaction

CH 3CH 2OH  NAD CH 3CHO  NADH  H 

ADH

A  B enzyme
 P  Q (12)

The possible enzyme-substrate are as follows

EAB, EPQ, EBQ, as ternary complexes

EA, EB, EP, EQ as binary complexes

Most two substrate reactions are grouped under two headings

a. Single displacement reactions

b. Double displacement reactions

(a) Single displacement reactions

Reaction of A and B as substrates must have both A and B attached to catalyst to form EAB.

This may happen as random order or ordered order.

In random order we have the following

E  A  EA (12)

EA  B  EAB (13)

Or we may have the following arrangement

E  B  EB (14)

EB  B  EAB (15)

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In ordered single displacement reaction we have the following

E  A  EA (16)

EA  B  EAB (17)

This means that A is the leading substrate

Many dehydrogenases reactions which use NAD as cosubstrate are good examples of ordered
reactions such as as follows

Malate  NAD  MDH

 Oxaloacetate  NADH  H  (18)

The major steps in the above reaction are as follows

 E  NAD 
NAD  E  (complex) (19a)

E  NAD   Malate 
 E  NAD  Malate (Ternary complex) (19b)

 Oxalate NADH  H 
E  NAD  Malate  MDH  (19c)

The reaction is therefore ordered

(b) Doble displacement reaction include the following

A E 
 EA  P (20)

 Q  NADH  H  (Amino transferase class of enzymes)

EA  NAD  (21)

18.5.4 Effect of pH on Enzyme Kinetics

Assumptions are as follows:

1. That amino acids change charge as pH is changed

2. That only effect of pH on Vmax is considered
3. Effects on Km are negligible
4. That the pH will not change the limiting step
5. That substrate is saturating

Consider also that:

a. -E- is the active form of single ionising chain in the enzyme

b. EH+ is inactive form of enzyme
c. E2- is inactive form of enzyme

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  EH  Ka
1
 E   H  Ka
2 E 2  H 
 (22)
Inactive active Inactive

The acid dissociation constant is given by the following

EH 
Ka  (23)
EH

EH 
EH  (24)
Ka

The fraction f of the enzyme in active unprotonated form is given by

E Ka
f  
 (25)
E  EH Ka  H 

If the Vmax is the maximum rate which can occur for the enzyme in the unprotonated form, then at any
pH Vmax is given by the following

Vmax  (Vmax ) m f (26)

Ka
Vmax  (Vmax ) m (27)
Ka  H 

Similarly for the second ionizing group which deactivates the active site, the effect of Vmax at any pH is
given by the following:

(Vmax ) m
Vmax  (28)
H  Ka2
1 
K a1 H 

18.5.5 Effect of Temperature on Enzyme Kinetics

Rate of enzyme catalysed reactions increases with temperature

One can therefore predict the activity of the reaction at any temperature

However the rate of enzyme catalyzed reaction increases to a maximum and then decreases as
the protein is denatured by heat.

The temperature affects the activity of the enzyme as well as its stability

For most simple reactions the activation energy is 300MJ/kmol or less

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For very fast reactions the activation energy is very low and are less affected by temperature.

For typical biochemical reactions the activation energy is around 50MJ/kmol

An increase in temperature from 25-35oC doubles the rate of reaction

However for activation energies of 85MJ/kmol, the rate can triple over the same temperature
range of the reaction

The ratio of rate constants of reaction at two temperatures 10oC apart is called Q10 value of
the reaction

The Q10 value of the reaction ranges from 1.7 – 2.5 in enzyme catalysed reactions

The Arrhenius expression is given as follows

k  Ae  E / RT (29)

In biochemical reactions k is replaced by kcat at constant enzyme concentration

The total concentration of enzyme (Etot) is directly related to Vmax and the activation energy E is replaced
by Ea and (29) becomes (30)

Vmax  Ae  Ea / RT (30)

A plot of Vmax verses 1/T gives a straight line with a slope of -Ea/R

The activation energy can thus be determined

The effect of temperature denatures protein thus affecting the enzyme

Deactivation of enzyme is the effect of temperature on the protein

The deactivation of protein by temperature is a first order decay process

Consider the deactivation of active enzyme Eact to inactive form E1 which can be described by the first
order rate constant kde

Eact 
kde
E1

dEact
  k de Eact (31)
dt

Integrating (31) we have the following

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 dEact
Eact ( t ) t
Eact ( 0 ) E
act
   kde dt
0
(32)

Eact
In  kde dt (33)
Eact

Eact (t )  Eact ( 0) e  kdet (34)

The half life can thus be determine bysetting the enzyme activity at half its initial value as follows

Eact ( 0 )
Eact (t )   k de dt (35)
2

When we substitute 35 into 34 we get the following

t
Eact ( 0)  k de
 Eact ( 0) e 2
(36)
2
t
1  k de
e 2 (37)
2

In 2
t1/ 2  (38)
k de

18.6 EXERCISES
Q 18.6.1

Discuss the activities of enzymes and the effects of inhibition on the reactions

Q18.6.2

What are the effects of pH on enzymatic reactions?

Q 18.6.3

Describe in detail the relationship between enzyme reactions and the temperature

Q 18.6.4

As engineers in a process plant explain to the management the problems associated with the
use of enzymes in enhancing the biochemical reaction?

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18.7 REFERENCES
1. Coulson J M and Richardson J. F (1991). Chemical Engineering: Chemical and Biochemical
Reactors and Process Control; Butterworth Heinemann
2. Fogler, H. S. (2016). Elements of Chemical Reaction Engineering, 5th Edition, Prentice Hall
International Inc.
3. Levenspiel. O. 1998. Chemical Reaction Engineering, 3rd Editon, Wiley.
4. Schmidt, L. D (2004), “The Engineering of Chemical Reactions”, 2nd Edition, Oxford University
Press, Oxford.
5. Froment, G. F., Bischoff, K. B. and De Wilde, J., (2010). “Chemical Reactor Analysis and design”,
3rd Edition, Wiley, New York.
6. Cooper A. R.and Jeffreys, G V., (1971). “Chemical Kinetics and Reactor Design”, Oliver and
Boyed, New York.
7. Relclaitis, G. V. (1983), “Introduction to Materials and Energy Balances”, 1st Edition, Wiley, New
York.

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