Department of Chemical and

Environmental Engineering
CHEE3024 BIOCHEMICAL ENGINEERING

Chapter 2:
Kinetics of Enzymatic Reactions
 Learning Outcomes

1. Understand and describe the basics of microorganisms.

2. Demonstrate a working knowledge on enzyme applications in

industries, and its associated kinetics models.

3. Understand and apply the principles of cell growth kinetics,

both theoretically and mathematically.

4. Identify and apply knowledge on industrial bioprocess designs.

5. Utilise mathematical skills to determine energy requirements

and biomass yields.

6. Demonstrate the ability to critically analyse ethical issues

surrounding biotechnology advancements.
2
 Overview
1. Introduction to Kinetics
2. Order of Reaction
3. Reaction Yield
4. Factors Affecting Kinetics

3
 1. Introduction to Kinetics

The study of:

the rate of change of reactants to products

Velocity of reaction can be expressed in different ways…

A ↔ P
dA n dP
− = k[ A] =
dt dt
Σ quantity
Rate =
d (time )
4
The Definition of Reaction Velocity

5
 2. Order of Reaction

When the progress of a reaction such as

aA + bB ↔ products

is measured experimentally, the rate can be
approximately expressed by
α β
− rA = kCA CB

where the rate constant is only dependant on
temperature.
6
 2. Order of Reaction (cont’d)
α β
− rA = kCA CB

The reaction is said to be αth order with respect to A and
βth order with respect to B. Overall order is (α+β)th order.

α and β are not necessarily equal to the stoichiometric
coefficients a and b

α and β may be negative or positive, integers or fractions

7
 2. Order of Reaction (cont’d)

the unit of k must be expressed precisely, using the
particular values of α and β.

e.g. if -rA = kCA2CB (unit of C: kmol/m3)

(kmol 3 )
units of k = m s
(kmol 3 ) 3
m
6
m
= 2
kmol .s

8
 First Order Reactions

Many bioreactions are 1st order, such as the clearance of

drugs from blood by peripheral tissues.

E.g. the rate of breakdown of A, at a given temperature,
is proportional to the concentration of A

A → products
3
− rA = kCA (kmol / m s)

9

consider a batch of material which is not being replenished with
fresh A.

At any time t…

dC A
−r = −
dt

− r = kC A

dC A
= −kdt
CA
10
 First Order Reactions (cont’d)

say that initial conditions are t = 0 and CA=CAo
CA t

then… dCA
∫C CA = ∫0 − k.dt
A0

[ln C A ]CC AA0 = [− kt ]t0

∴ ln C A − ln C A0 = − kt L (1)
CA
ln = − kt K (2)
C A0

Alternatively,
− kt
C A = C A0 e
11
 First Order Reactions (cont’d)

from equation (1) a plot of lnCA against t will give a straight line
of slope –k

lnCA

ln C A − ln C A0 = − kt

time
12
 First Order Reactions (cont’d)

Equation (2) shows an exponential decrease in
reactant concentration as a function of time.
Conc. CA

CAo
CA
ln = − kt
CAo/2
C A0
CAo/4

Time

13

From equation (2), it is clear that in all parts of the
decay curve, the time taken for the concentrations to
drop by a certain fraction is constant.

The ‘half-life’ of the reaction is commonly quoted, i.e.

the time taken for the concentration to fall by one half.

14
 CA
ln = − kt
C A0
if C A0 = 2 × C A then
ln 2
half life, t 1 = K (3)
2 k

Remember

Half Life (same as doubling time)
0.693
t1 =
2 k

15
 Second Order Reactions

2nd order reactions like hydrolysis can be made to appear
as pseudo 1st order reactions if the water is in excess.
• Why??
k2

Reactions are reversible, but the product can go onto

another reaction, hence reversibility is less likely.

16
 3. Reaction Yield

“the extent to which reactants are converted to
products”
or more generally…

“the amount of product formed per amount of

reactant provided”

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 3. Reaction Yield (cont’d)

When reactants or products are involved in additional
reactions, the observed yield may be different from the
theoretical yield.

Enzyme x B
A
Enzyme y
C
mass / mole of product present
Observed yield =
total mass / mole of reactant consumed

18
 4. Factors Affecting Kinetics

We are going to look at three factors affecting the
reaction velocity.
1. Temperature
2. pH
3. The relationship between Enzymes and Substrate

Michaelis-Menten equation

Vmax and Km – How to determine?

19
 Temperature

Arrhenius was a scientist in the 19th century.

Recognised the relationship between reaction rate and

the temperature at which the reaction is carried out.

An equation to approximate the variation of rate constant

k with temperature was established in 1889.

20
 Temperature

− Ea
k = A.e RT

where A and Ea are constants

R: Universal gas constant (8.314 kJ/kmol.K)

T: absolute temperature

A: pre-exponential constant

must have the same units as k (the reaction rate constant)

Ea must have the same units as RT, e.g. kJ/kmol and is call
the “energy of activation”
21
 Experimental Data

To test experimental results
take logs, i.e.
lnk
k − Ea
ln =
A RT

A plot of lnk against 1/T will

have a slope equal to -Ea/R
and an intercept of lnA

1/T

22
 Temperature and Enzyme Based Reactions

Plots of reaction velocity vs. temperature for most

enzymes give bell shaped curves

The optimum
temperature is generally
around 40 °C to 45 °C for
enzymes from mammals.

Reaction rates generally
double for every 10 °C
rise.

Denaturation occurs
once temperatures
exceed a critical limit:

thermal deactivation
occurs, and the velocity
quickly drops.

23
 pH

As with temperature, reactions
show a bell-shaped curve with pH
changes.

If the reaction environment is not at

the correct pH range, there is a high
possibility that the enzyme in
question will not act as a ‘catalyst’ at
all.

This is due to the poor binding

abilities of the enzyme when the pH
is unfavourable.

24
25 Thursday, March 28, 2019

H83BCE Biochemical Engineering

Lecture 2:
Part II
Kinetics of Enzymatic Reactions
 The Relationship between Enzyme and
Substrate
1. Relationship between initial velocity, Vo, and substrate
concentration, [S]
(a) Michaelis-Menten equation
(b) Vmax and Km – How to determine?

2. Enzyme inhibition
(a) Competitive
(b) Uncompetitive
(c) Non-competitive

26
 Relationship between Initial Velocity and Substrate
Concentration

Henri in 1903 looked at the relationship of velocity and substrate
concentration.

Michaelis and Menten in 1913, published their findings on the

relationship of initial velocity, vo and [S].

27
 [E] & [S] Relationship

In general the following relationship exists
k1 k3
[E] + [S] k2
[ES] k4
[E] + [P]

Where one substrate is bound to one enzyme.

If the initial reaction is considered, [P] is small, thus
P
ES can be written off.
k1 k3
[E] + [S] [ES] [E] + [P]
k2

Therefore…
28

The rate of formation of [ES] = k1[E][S] (at time t)

Equally, the rate of formation of [E] and [S] from [ES] = k2[ES]
Reverse reaction

Michaelis-Menten assumption:

the equilibrium between [E]+[S] ↔ [ES] is instantaneous and maintained
throughout the process.

Therefore…
k1[E][S] = k2[ES] + k3[ES]
k1[E][S] = [ES].(k2 + k3)
[E ][S ] = (k2 + k3 ) = K Michaelis
[ES ] k1
m Constant

k1 k3
[E] + [S] [ES] [E] + [P]
k2 29

At any time t, some enzymes are engaged in a reaction

Therefore…[E] = [Eo]-[ES]

[Eo] is the concentration of enzyme before the process starts.

∴sub. for term [E] into eqn. on previous slide:

[E] ([Eo ] − [ ES ]).[S ] = K

[ES ] m

[ Eo ].[ S ]
[ ES ] =
[S ] + K m

Initial Velocity Vo=k3[ES]

∴sub. for term [ES] using eqn. above:

k3[ Eo ].[ S ] [ES]
vo =
[S ] + K m
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The maximum velocity of the reaction is when all the enzymes are engaged:
Vmax = k3[Eo] i.e. [ES] = [Eo]

Vmax .[ S ]
vo =
[S ] + Km

Since [S] is normally >> [E], [S] at any time during the reaction approximates to
[So]

Therefore…

Vmax .[ So ]
vo =
[ So ] + K m

This is the Michaelis-Menten Equation

This assumes that k2 >> k3

31
 What does it all mean?

However, when [S] is small compared to Km, then:

Vmax .[ S o ]
vo =
Km
Why does Vmax/2 correspond to Km?

The reaction rate is proportional to the substrate

concentration. When [S] = Km:

Vo Vmax .[ S o ]
vo =
[ So ] + [So ]

when the substrate concentration
is high compared to Km, V Vmax
approaches Vmax as Km becomes =
negligible: 2

Consequently, Km is the substrate concentration
Vmax .[ S o ] which gives half the maximum rate.
vo = = Vmax
[ So ]
Which also means that Km has the same units as
[So].

32

The relationship between enzymes, substrates and products
can be a little more complicated than the initial reaction
process.

E.g. the transformation of reactants to products whilst
attached to the enzyme (i.e. [ES]  [EP]) is neglected.
k1 k3 k4
[E] + [S] [ES] [EP] [E] + [P]
k2

If k3 (in this case) is << k2, k1 and k4,then,

k3 can also be known as kcat which is the
TURNOVER NUMBER
number of substrate molecules converted to product molecules
turnover no. =
enzyme ⋅ time

Therefore, Vmax = kcat[Eo]

33
 The Importance of Km
(k 2 + k3 ) = K
m
k1

It is logical that if Km is big, then k1 is < k2+k3. Therefore big Km values mean poor binding. Small Km
values mean stronger binding.

E.g.: Some Chinese and Japanese get pronounced facial flushing from smaller amounts of alcohol.

This is called Vasodilation and results from enhanced levels of ethanal (CH3CHO) in the body.

CH3CH2OH CH3CHO CH3COOH

Consider the conversion of CH3CHO to CH3COOH:

k1 k3
[CH3CHO] + [E] [CH3CHO.E] [CH3COOH.E]
k2

The enzyme in some Asians has a high Km for transformation of CH3CHO to CH3COOH

therefore a higher steady state of CH3CHO results.

High Km ~ small k1, i.e. poor binding of E to CH3CHO substrates.

CH3CHO accumulates after drinking, leading to severe hangover symptoms.
34
 How to Find Vmax and Km?

Since Velocity approaches Vmax asymptotically, it is not possible to
calculate Km using Vmax/2.

Lineweaver and Burk in 1934 inverted the MM equation:

1 [ So ] + K m Km 1 1
= = . +
vo Vmax [ So ] Vmax [ So ] Vmax

35
The Lineweaver-Burk (LB) Plot

1 Km 1 1
= . +
vo Vmax [ S o ] Vmax
36
 Criticisms of the LB Plot

1. It is difficult to guess how much graph paper is needed on the

other side of the y-axis to reach the x-axis.

2. It allows low concentration of [S] values to be used which can

skew the plot.

3. Deviations from linearity are less easy to spot compared with

other means of interpretation.

37
 The Eadie Hofstee (EH) plot

Uses the LB plot but multiplies every term by vo.Vmax.

vo
vo = − K m . + Vmax
[ So ]

A plot of Vo (y-axis) against Vo/So (x-axis) should yield a straight line

with intercept Vmax and slope –Km.

38
 The EH plot

vo
vo = − K m . + Vmax
[ So ]
39
 The Hanes Plot

Multiplies every term in LB equation by [So]

[ So ] 1 Km
= .[ So ] +
vo Vmax Vmax

A plot of [So]/vo against [So] gives a straight line with intercept

Km/Vmax and slope 1/Vmax

40
 The Hanes Plot

[ So ] 1 Km
= .[ So ] +
vo Vmax Vmax

41
 Comparison of the LB, EH and Hanes Models

LB model is generally used by the majority of enzymologists.

Enzymologists: biochemical nature & activity of enzymes.

EH and Hanes models are preferred by enzyme kineticists.

Enzyme kineticists: rate of change in concs. Of reactants & products in a
chemical rxn.

42
 Summary

Key points to remember:

Enzymes

Kinetics

Velocity of reaction

Reaction order (zero, first, second, pseudo-first)

Half-life

Reaction yield (theoretical vs. observed)

Arrhenius equation

Relationship between pH and enzyme

Michaelis-Menten equation

Vmax and Km

Turnover number

Importance of Km

Lineweaver-Burk plot

Eadie Hoftstee plot

Hanes plot
43
 CHEE 3024 Biochemical Engineering
Lecture 2: Kinetics of Enzymatic Reactions
Part III:
Inhibited Enzyme Kinetics
 Enzyme Inhibition

Inhibitors:

Substances that decrease the rate of an enzyme-catalysed reaction.

There are two modes of inhibition:

Reversible

May dissociate more easily from the enzyme after binding.

Non-Reversible

E.g. heavy metals (lead, mercury etc.) – form a stable complex with
enzyme and reduce enzyme activity.

May be reversed only by using chelating agents, e.g. EDTA
(ethylenediaminetetraacetic acid) and citrate.

45
 Supplementary info

Reversible reaction:

Reacts with enzymes and change it chemically.

E.g. via covalent-bond formation.

Chelating agent:
• a substance whose molecules can form several bonds to a single ion.
• E.g. ethylenediamine ligand (shown in figure).

46
 Types of enzyme inhibition
1) Enzyme inhibition
a) Competitive
b) Uncompetitive
c) Non-competitive

2) Substrate may act as inhibitor in some cases.

47
Competitive Inhibition

The effect of a reversible
inhibitor will become evident
quite rapidly as a reaction is
taking place.

We shall look at three cases

of reversible inhibition in
more detail although there
are more types.

48
 Competitive Inhibition (1)

An inhibitor that competes for the same site on the
enzyme.

Same site

49
 Competitive inhibition (2)

Inhibitor bonds to another site, invalidating another site
for the substrate.

Different site

50
 Inhibition of Product Formation – Competitive Inhibition

This increases Km for the reaction, but not the Vmax.

On the LB Plot the change in the graph is as follows:

I>0

I=0

Can be overcome by high concentrations of substrate.

51
 Supplementary info: Competitive inhibition

Vmax constant.

Add more substrate to out-compete the inhibitor.

Km,I will increase, as it takes more substrate (i.e. higher

concentration of S) to reach the Km point that corresponds to ½
Vmax.

I often similar structure to S.

52
 Uncompetitive Inhibition

Uncompetitive inhibitors bind only to [ES] rather than free enzymes.

Reduction in both Vm and Km.

Vmax is decreased by some conversion of [ES] to inactive [ESI].

Since it is the ES that the inhibitor attacks, adding more [S] will not solve the
problem.

Km also decreases because the equilibria is shifted in favour of [ESI] complex
formation rather than product or free [E] or [S].

Reduction in Vm has a more pronounced effect than the reduction

in Km:

Net result: reduction in reaction rate.

53
Uncompetitive Inhibition

54
 Inhibition of Product Formation – Uncompetitive Inhibition

LB Plot:

I>0

I=0

55
 Non-Competitive Inhibition

• Inhibitors are not substrate analogs.

56
 Inhibition of Product Formation – Non-competitive
Inhibition

The LB plot changes somewhat.

Km is the same as before but Vmax is lowered.

High substrate concentration would not overcome noncompetitive inhibition.

Other reagents need to be added to block binding of the inhibitor to the enzyme.

I>0

Overall, this result is rare.

I=0

57
 Illustration:
Competitive, uncompetitive and non-competitive inhibitions

58
 Supplementary info

Non-
Competitive Uncompetitive
competitive
• Binds to E, not ES. • Binds to ES. • I has identical affinity
• Increases Km: • Reduces Km, reduces for both E & ES.
• I interferes with S- Vmax. • Km constant:
binding. • I affects S-binding by: • Doesn’t affect S-
• Vmax constant: • Increases E’s affinity binding.
• I doesn’t hamper ES for S (hence reduces • Reduces Vmax:
catalysis, as I cannot Km). • EI binding hampers
bind to ES. • I affects catalysis catalysis.
(hence reduces Vmax).

59
 Substrate inhibition and its applications

Definition: At high [S], there is a progressive decrease in activity.

This may indicate the existence of 2 S-binding sites in E.

However, only 1 site leads to catalysis.

At low S:

High-affinity site occupied – normal kinetics follow.

At high S:

Second site on E (inhibitive site) becomes occupied, thereby inhibiting E.

Applications: drugs.

I has strikingly similar structure to S.

E.g. HIV drugs: ritonavir  resembles protein that is S for HIV protease.

60
 Summary

Key points to remember:

Competitive enzyme inhibition

same/different site Influence on

Uncompetitive enzyme inhibition Vmax and Km

Non-competitive enzyme inhibition

61