02.enzyme Kinetics

Adama Science &
Technology University
Department of Chemical
Engineering
Chapter Two
02. Enzyme Kinetics

February 26, 2021 Slide By Eba A
Adapted from James Lee, 2nd Edn
1
Course Contents
1. Biotechnology and Biochemical
2. Enzyme Kinetics
3. Immobilized Enzyme
4. Industrial Applications of Enzymes
5. Cell Kinetics and Fermenter Design
6. Sterilization
7. Agitation and Aeration
8.Downstream Processing

2
02. Enzyme Kinetics
Enzymes
• Are biological catalysts that are protein
molecules in nature.
• Are produced by living cells (animal,
plant, and microorganism)
• Catalyze the making and breaking of
chemical bonds at a small portion of
their surface, which is known as the
active site.
• Increase the rate of reaction without
themselves undergoing permanent
chemical changes.
3
02 Enzyme Kinetics Cont‟d
Enzyme reactions are different from
chemical reaction in the following
ways.
Highly specific enzyme catalyst, and
catalyzes only one reaction.
Much faster reaction rate
Mild reaction conditions(lower T,
pH)
Sensitivity or unstablity of enzyme
molecules
4
Nomenclature of Enzymes
• Originally named arbitrarily
rennin curding of milk to start cheese-
making process
pepsin hydrolyzes proteins at acidic
pH
trypsin hydrolyzes proteins at mild
alkaline pH
• Naming was later improved by
introducing the suffix „ase‟
5
Nomenclature of Enzymes…
 Name of substrate + ase
Enzyme Substrate Products
Alpha amylase starch Glucose. Maltose and

oligosaccharides
lactase Lactose Glucose & galactose
Lipase Fat Fatty acid, glycerol
maltase maltose glucose
urease Urea Ammonia , Carbondioxide
cellobiase cellobiose glucose

6
Reaction which is catalyzed + ase
Enzyme Reactions Products
Alcohol Ethanol + NAD+ Acetaldehyde +

dehydrogenase NADH2
Glucose isomerase Glucose Fructose
Glucose Oxidase D-glucose + Gluconic acid

O2+H2O
Lactic acid Lactic acid Pyruvic Acid
Dehydrogenase
7
• International Enzyme Commission
developed the new system categorizes
all enzymes into six major classes in
1964.
• Oxidoreductase . Ligases
• Transferase
• Hydrolases
• Lyases
• Isomerases
8
Commercial Application of Enzym
•Enzymes have been utilized
commercially since the 1890s.
•Fungal cell extracts were first
added to brewing vats to facilitate
the breakdown of starch into
sugars.
•The fungal amylase takadiastase was
employed as a digestive aid in the
United States as early as 1894.
9
• The enzymes produced commercially can be
classified into three major categories
1. Industrial enzymes, such as amylases,
proteases, glucose isomerase, lipase,
catalases, and penicillin acylases
2. Analytical enzymes, such as glucose
oxidase, galactose oxidase, alcohol
dehydrogenase, hexokinase, muramidase,
and cholesterol oxidase
3. Medical enzymes, such as asparaginase,
proteases, lipases, and streptokinase
10
• alpha-amylase, glucoamylase, and glucose
isomerase serve mainly to convert starch
into high-fructose corn syrup (HFCS), as
follows: A- Gluco
amylase amylase
Corn Starch Thinned
Glucose
Starch
Glucose
Isomerase
HFCS
HFCS is used in soft drink production

11
•Alkaline protease is added to
laundry detergents as a cleaning aid
•Proteins often precipitate on soiled
clothes or make dirt adhere to the
textile fibers.
•The stain can be dissolved easily by
addition of protease to the
detergent
12
Simple Enzyme Kinetics
Enzyme kinetics
• deals with the rate of enzyme reaction and
how it is affected by various chemical and
physical conditions.
Kinetic studies
• Provide information about the basic
mechanism of the enzyme reaction and
other parameters that characterize the
properties of the enzyme.
• Yields rate equations for calculating reaction
time, yields, and optimum economic
condition, which are important in the design
of an effective bioreactor.
13
• Assume that a substrate (S) is converted to a
product (P) with the help of an enzyme (E) in a
reactor as
S E P
• 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.
• The rate of reaction can be expressed in
terms of either the change of the substrate Cs
or the product concentrations Cp as follows:
𝒅𝑪𝒔 𝒅𝑪𝒑
𝒓𝒔 = − Or 𝒓𝒔 =
𝒅𝒕 𝒅𝒕
14
 The change of product and  The effect of substrate

substrate concentrations with concentration on the initial reaction
respect to time rate.
 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 in the next slide.

15
Reaction velocity

16
Enzyme Kinetics
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 Vmax is proportional to
the enzyme concentration within the range of the
enzyme tested.

17
• Henri observed this behavior in 1902 and
proposed the rate equation
Vm [ S ]
v 
Km  [ S ]
• where Vmax and KM are kinetic parameters
which need to be experimentally determined.
• The equation expresses the three preceding
observations fairly well.
• The kinetic mechanisms which support the
above equation should be determined
18
• 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
E + S ‹--------› ES
k1
k2
ES --------› E + P k3

19
Enzyme Kinetics
• The “lock and key” theory accounts for
the formation of the enzyme-substrate
complex
• There is a topographical, structural
compatibility between an enzyme and a
substrate which optimally favors the
recognition of the substrate.

20
Assumptions for rate Eqn
1. The total enzyme concentration stays
constant during the reaction, that is,
[ Eo]  [ E]  [ ES ]
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.

21
Rate Equations
In addition to the preceding assumptions,
there are three different approaches to
derive the rate equation:
1. Michaelis-Menten approach (Michaelis
and Menten, 1913)
2. Briggs-Haldane approach (Briggs and
Haldane, 1925):
3. Numerical solution:

22
Michaelis-Menten approach
• The product-releasing step is much slower
than the reversible reaction and the slow
step determines the rate, while the other
is at equilibrium.
• The first step for an enzyme reaction is
based on a very weak interaction.
• Therefore, it is reasonable to assume that
the enzyme-substrate complex formation
step is much faster than the product
releasing step which involves chemical
changes.
23
• If the slower reaction determines the overall
rate of reaction, then the rate of product
formation:
d [ P]
v  d [ S ] / dt  k3 [ ES ]
dt
• The concentration of the enzyme-substrate

complex [ES], can be related to the substrate
concentration [S] and the free-enzyme
concentration [E] from the assumption that
the first reversible reaction is in equilibrium.
Then, the forward reaction is equal to the
reverse reaction so that
K1[E][S]=K2[ES] [ Eo]  [ E]  [ ES ]
24
• The equilibrium constant can be expressed by the
following equation in a dilute system.

k1
k3
E+S ES PE
k2
k 2 [ E ][ S ]
Km  
k1 [ ES ]

25
• Then rearrange the above equation,
𝐸 [𝑆]
[ES]= 𝐾𝑚
• Substituting [E] in the above equation with enzyme

mass conservation equation
[ E ]  [ E0 ]  [ ES ]
yields, ([ E 0 ]  [ ES ])[ S ]
[ ES ] 
Km

26
• [ES] can be expressed in terms of [S],
[ E 0 ][ S ]
[ ES ] 
K m  [S ]
• Then the rate of production formation v can
be expressed in terms of [S],
d [ P] k 3 [ E 0 ][ S ] Vm [ S ]
v  k 3 [ ES ]  
dt K m  [S ] K m  [S ]
Where Vm  k 3 [ E 0 ] represents the maximum forward
reaction
27
Defn of Michael‟s Parameters
Km
• is often called the Michaelis-Menten
constant.
• corresponds to the substrate
concentration, giving the half maximum
reaction velocity.
• is equal to the dissociation constant K1
or the reciprocal of equilibrium constant
Keq as
Km=k2/k1=K1= [S][E]/[ES]=1/Keq
28
• The unit of Km is the same as that of [S].
• When Km is equal to [S], V is equal to one half
of Vmax according
• Therefore, the value of Km is equal to the
substrate concentration when the reaction
rate is half of the maximum rate Vmax
• Km is an important kinetic parameter because
it characterizes the interaction of an enzyme
with a given substrate.
• Small Km means tight binding; high Km means
weak binding
29
Vmax( Maximum Reaction velocity)
 is proportional to the initial enzyme
concentration(Vm=k3[Eo]).
 has a unit of kmole/m3s.
 Vm= 2V when Km=[S]
ES remains constant while S is converted to P. Here

all the substrate is converted to P
30
Briggs-Haldane Approach
• The change of the intermediate concentration
with respect to time is assumed to be
negligible, that is, d [ES]/dt =0.
• known as the pseudo-steady-state (or quasi-
steady-state) assumption in chemical kinetics.
• The initial substrate concentration [S0] greatly
exceeds the initial enzyme concentration [E0],
since [E0] was small.
• It is shown that in a closed system the quasi-
steady-state hypothesis is valid after a brief
transient if [S0]>> [E0].
31
• With such assumption, the equation
representing the accumulation of [ES]
becomes
d [ ES ]
 k1 [ E ][ S ]  k 2 [ ES ]  k 3 [ ES ]  0
dt
Solving this algebraic equation yields
k1 [ E ][ S ]
[ ES ] 
k 2  k3
32
Substituting the enzyme mass conservation
equation
[ E ]  [ E0 ]  [ ES ]
in the above equation yields
k1 ([ E 0 ]  [ ES ])[ S ]
[ ES ] 
k 2  k3
Using [S] to represent [ES] yields

[ E 0 ][ S ]
[ ES ] 
k 2  k3
 [S ]
k1

33
• Then the product formation rate becomes
d[ P] k 3 [ E 0 ][ S ]
v   k [ ES ] 
dt 3 k 2  k3
 [S ]
k1
Then Vm [ S ]
v
K m  [S ]
Where Vm  k [ E 0 ] same as that for rapid equilibrium assumption.

3
k 2  k3
Km 
Where k1

34
Comparison of Approaches
Michaelis-Menten Briggs-Haldane
Assumption: d[ES]/dt ≈ 0
k 1 [E][S]  k 2 [ES]
Equation: Vm [S] Vm [S]

v  v 
K m  [S] K m  [S]
Maximum
forward
reaction rate:
Vm  k [E 0 ] Vm  k 3 [E 0 ]
3
k2
k2  k3
Constant: Km  Km 
k1 k1

35
Hands on Exercise -1
When glucose is converted to fructose by
glucose isomerase, the slow product formation
step is also reversible as:
k1 k3
S+E ES ES P +E
k2 k4
• Derive the rate equation by employing

i. the Michaelis-Menten an
ii. the Briggs-Haldane approach.
iii. Explain when the rate equation derived by
the Briggs-Haldane approach can be
simplified to that derived by the Michaelis-
Menten approach.
36
Numerical Solution
• Three rate equations can be written for [E]
[ES] and [S] as
d[P]/dt =k3[ES]
d[ES]/dt =k1[S][E]-k2[ES]-k3[ES]
d[S]/dt =-k1[S][E]+k2[ES]
• These three equation can be solved
simultaneously without simplification.
• Since the analytical solution of the preceding
simultaneous differential equations are not
possible, we need to solve them numerically by
using a computer
37
Evaluations of Kinetic Parameters
1. Make a series of batch runs with
different levels of substrate
concentration.
2. Calculate initial reaction rate as a
function of initial substrate
concentrations.
3. Plot the results graphically so that the
validity of the kinetic model can be
tested and the values of the kinetic
parameters can be estimated.
38
Evaluations of Kinetic Parameters
• plot V against [S]
• The asymptote for V will be Vmax and Km
is equal to [S] when V= 0.5 Vmax.
• Estimation of Km and Vm will be difficult as
determination of asymptote is impossible
• Thus, Michael-Menten equation should be
rearranged and plotted.
• This can be achieved in three ways:

39
Lineweaver-Burk Plot
Vm [ S ]
v
K m  [S ]
Linearizing it in double-reciprocal form:
1 1 Km 1
 
v Vm Vm S
• A plot of 1/V versus 1/[S] will give a straight line with
slope of Km/Vmax and y intercept of 1/Vmax
• Such a plot is known as a Lineweaver-Burk double
reciprocal plot
40
Lineweaver-Burk Plot
• It is difficult to determine Vmax
experimentally
• The equation for a hyperbola can
be transformed into the equation
for a straight line by taking the
reciprocal of each side
• The formula for a straight line is y
= mx + b
• A slope equals to Km/Vm
• y-intercept is 1/Vm.
• 1/v approaches infinity as [S] decreases
• More often employed

41
Eadie-Hofstee Plot
• gives slightly better v
weighting of the data than v  Vm  K m
the Lineweaver-Burk plot [S ]
• disadvantage of this plot is
that the rate of reaction V
appears in both coordinates
while it is usually regarded as
a dependent variable.
• The slope is –Km

• y-intercept is Vm.
42
Hanes-Woolf (Langmuir) Plot
• Based on the data [S ] K m
 
1
[S ]
distribution, the v Vm Vm
Langmuir plot ([S]/V

versus [S]) is the most
satisfactory of the
three, since the points
are equally spaced.
• The slope is 1/Vm
• y-axis intercept is
Km/Vm
43
Summary,
• In conclusion, the values of the Michaelis-
Menten kinetic parameters, Vmax and Km, can be
estimated, as follows:
 Make a series of batch runs with different levels
of substrate concentration at a constant initial
enzyme concentration and measure the change
of product or substrate concentration with
respect to time.
 Estimate the initial rate of reaction from the [S]
or[P] versus time curves for different initial
substrate concentrations.
 Estimate the kinetic parameters by plotting one
of the three plots explained in this section.
44
Hands on Exercise-2
From a series of batch runs with a constant
enzyme concentration, the following initial
rate data were obtained as a function of
initial substrate concentration.
Substrate Conc (mmol/L) Initial reaction Rate (mmol/min)
1 0.2
2 0.22
3 0.3
5 0.45
7 0.41
10 0.50
15 0.40
20 0.33

45
Hands on Exercise-2…
a. Evaluate the Michaelis-Menten kinetic
parameters by employing the Langmuir plot,
the Lineweaver-Burk plot, the Eadie-Hofstee
plot, and nonlinear regression technique. In
evaluating the kinetic parameters, do not
include data points which deviate
systematically from the Michaelis-Menten
model and explain the reason for the
deviation.
b. Compare the predictions from each method
by plotting V versus [S] curves with the data
points, and discuss the strengths and
weaknesses of each method.
c. Repeat part (a) by using all data.
46
Bioreactors with Simple Kinetics
• A bioreactor is a device within which biochemical
transformations are caused by the action of
enzymes or living cells.
• The bioreactor is frequently called a fermenter
whether the transformation is carried out by
living cells or enzyme.
• The simplest reactor configuration for any
enzyme reaction is the batch mode.
47
Batch Reactor
• A batch enzyme reactor is normally
equipped with an agitator to mix the
reactant, and the pH of the reactant is
maintained by employing either a buffer
solution or a pH controller.
• An ideal batch reactor is assumed to be
well mixed so that the contents are
uniform in composition at all times.
• Assuming Michaels Menten Equation is
𝑑𝑆 𝑉𝑚 𝑆
applicable − =
𝑑𝑡 𝐾𝑚+ 𝑆
48
Batch Reactor
• An equation expressing the change of the
substrate concentration with respect to
time can be obtained integrating the above
equation to get
𝑆𝑜
𝐾𝑚. 𝑙𝑛 + 𝑆𝑜 − 𝑆 = 𝑉𝑚. 𝑡
𝑆
This equation shows how [S] is changing with
respect to time. With known values of Vmax and
the change of [S] with time in a batch reactor can
be predicted from this equation.

49
Plug Flow Reactor
• In a plug-flow enzyme reactor (or tubular-
flow enzyme reactor), the substrate enters
one end of a cylindrical tube which is
packed with immobilized enzyme and the
product stream leaves at the other end.
• The properties of the flowing stream will
vary in both longitudinal and radial
directions(though the variation in the
radial direction is small compared to the
longitudinal direction)
50
Plug Flow Reactor
• The residence time of PFR is calculated
from
𝑆𝑜 − 𝑆 ln So
ln 𝑆𝑜 = −Km + Vm. t /( )
S
𝑆

51
CSTR for Substrate Conversion
• A continuous stirred-tank reactor (CSTR)
is an ideal reactor which is based on the
assumption that the reactor contents are
well mixed.
• Continuous operation of the enzyme
reactor can increase the productivity of
the reactor significantly by eliminating the
downtime.
• It is also easy to automate in order to
reduce labor costs.
52
CSTR for Substrate Conversion
• Substrate balance for CSTR
Input - Output + Generation
Accumulation
F[So]-F[S]+rsV=Vd[S]/dt
• For the steady-state CSTR, the
Substrate concentration is substrate concentration of the
now given by
𝜏 reactor should be constant,
[S]= -Km+Vmax[S]
{ 𝑆𝑜 − [𝑆]}
d[S]/dt=0
𝜏- residence time • Simplifying the previous equation
D- dilution rate
yields
F/V=D=Vmax[S]/{([So]-[S])(Km+[S]}

53
Inhibition of Enzyme Reactions
• Modulator (or effector) is a substance
which can combine with enzymes to
alter their catalytic activities.
• Inhibitor is a modulator which
decreases enzyme activity. It can
decrease the rate of reaction either
• competitively,
• noncompetitively, or
• partially competitively.
54
Inhibition of Enzyme Reactions
Inhibitors
are chemicals that reduce the
rate of enzymatic reactions
bind to an enzyme and
interferes with its activity
are usually specific and they
work at low concentrations
block the enzyme but they do
not usually destroy it
55
Reversible versus Irreversible
• Reversible inhibitors interact with an
enzyme via noncovalent associations
• Irreversible inhibitors interact with an
enzyme via covalent associations
• Reversible enzyme inhibition can be
classified as
Competitive inhibition
Noncompetitive inhibition
Uncompetitive inhibition
56
Competitive inhibition
• Competitive inhibitor competes with a
substrate for the enzyme - substrate binding
site
• There is a structural similarity between the
inhibitor and the substrate

57
Reaction Mechanisms
E+S ES E+P
+
I In competitive inhibition, the
inhibitor binds only to the free
enzyme, not to the ES complex
EI

58
Reaction Mechanism
•E + S
K1
K2
+
I
KI
EI
• Both the substrate and inhibitor compete for binding
to the same form of the enzyme: free form
• ESI complex is not formed

59
Competitive Inhibition
• The inhibition is most noticeable at
low [S] but can be overcome at
sufficiently high [S]
- Vmax remains unaffected
• Attaining Vmax requires higher [S] in
the presence of competitive inhibitor
- Apparent Km is increased

60
Competitive inhibitors alter the apparent
.
Km, not the Vmax

- Inhibitor
Vmax
Reaction Rate
+ Inhibitor Vmax,app = Vmax

Vmax Km,app > Km
2
Km Km,app
[Substrate]
61
The Lineweaver-Burk plot is diagnostic for
competitive inhibition
1 = Km,app 1
+ 1 Increasing [I]
v Vmax [S] Vmax
Km,app
1 Slope =
Vmax
v
1
Vmax
-1 1
Km,app
[S]
62
Relating the Michaelis-Menten equation, the v vs. [S] plot,
and the physical picture of competitive inhibition
Inhibitor competes .
with
substrate, decreasing - Inhibitor
its apparent affinity: Vmax
Reaction Rate
Km,app > Km
+ Inhibitor
Vmax
2
Km,app > Km
Formation of EI Vmax,app = Vmax
complex shifts reaction
to the left: Km,app > Km Km Km,app
[Substrate]

63
• Assume rapid equilibrium and with the
definition of

64
Competitive inhibition
• Simplifying
When [I]=0,
Remains that of Michaelis-menten equation

Is increased
65
Noncompetitive inhibition

66
Reaction Mechanism
E+S ES E+P
+ + In noncompetitive
inhibition, the inhibitor
I I binds enzyme regardless
of whether the substrate
is bound
EI + S ESI

67
km
•E + S
+ +
I I
kI km kI
EI+S ESI
• Since noncompetitive inhibitors do not interfere

in the binding of the substrate (the dissociation
constant of ES and ESI have the same value Km)
- Km is not affected
68
• However, increasing [S] can not abolish the
inhibition - (ESI) complex are formed and these
are incapable of progressing to reaction products
• The effect of a noncompetitive inhibitor is to
reduce [ES] that can advance to product
• Since Vmax = k3[Eo], and the concentration of
competent Eo is diminished by the amount of ESI
formed
- Vmax is decreased

69
Noncompetitive inhibitors decrease the
Vmax,app, but don‟t affect the Km
Vmax,app < Vmax

Km,app = Km

70
Why does Km,app = Km for noncompetitive
inhibition?
E+S ES E+P
+ + The inhibitor binds
equally well to free
I I enzyme and the ES
complex, so it doesn’t
alter apparent affinity
EI + S ESI of the enzyme for the

substrate
71
noncompetitive inhibition
1 = Km 1 1 Increasing [I]
+
v Vmax,app [S] Vmax,app
1 Slope =
Km
v Vmax,app
1
Vmax,app
-1 1
Km
[S]
72
Relating the Michaelis-
.
I I Menten equation, the v vs.

S [S] plot, and the physical
Enzyme S Enzyme
Inhibitor doesn’t interfere
picture of noncompetitive
with substrate binding,
Km,app = Km
inhibition
S
.
I I
Vmax - Inhibitor
S
Reaction Rate
Enzyme Enzyme
Vmax,app
1
V
+ Inhibitor
2 max
1
V Km,app V>max,app
2 max,app
Km < Vmax
Even at high
substrate levels,
Vmax,app K=m,app
Vmax= Km
inhibitor still binds,
Km Km,app
[E]t < [ES]
Vmax,app < Vmax [Substrate]

73
Assume
• Rapid equilibrium
• Same equilibrium constant of inhibitor
binding to E and ES……… KI
• Same equilibrium constant of inhibitor
binding to E and EI ……….Km

74

75
Remains in Michaelis-menten equation

Is reduced
When [I}= 0,
76
Uncompetitive Inhibition
E+S ES E+P
+ In uncompetitive
inhibition, the
I inhibitor binds
only to the ES
complex, it does
not bind to the
ESI free enzyme

77
Uncompetitive inhibition
• The ES complex dissociates the
substrate with a dissociation
constant equal to Km, whereas
the ESI complex does not
dissociate it (i.e. has a Km value
equal to zero)
- Km is decreased
• Increasing [S] leads to increasing [ESI] (a complex
incapable of progressing to reaction products),
therefore the inhibition can not be removed
- Vmax is decreased
Slide By Eba A Adapted from James Lee, 2nd
February 26, 2021 78
Edn
Uncompetitive inhibitors decrease both the
Vmax,app and the Km,app
Vmax,app < Vmax
Km,app < Km
Notice that at low substrate
concentrations,
uncompetitive inhibitors
have little effect on the
reaction rate because the
lower Km,app of the enzyme
offsets the decreased Vmax,app

79
uncompetitive inhibition
1 = Km,app 1 1
+
v Vmax,app [S] Vmax,app 1 Increasing [I]
=
Km 1
+
1 v
Vmax [S] Vmax,app
Km
Slope =
Vmax
1
Vmax,app
-1 1
Km,app
[S]
80
Relating the Michaelis-Menten equation, the v vs. [S] plot,
and the physical picture of uncompetitive inhibition
Enzyme .
Enzyme
S
S
Enzyme Vmax,app < Vmax

I
Inhibitor I
increases Km,app< Km
the amount of Enzyme Vmax - Inhibitor
Reaction Rate
enzyme bound
to substrate I S
Km,app < Km Vmax,app

1
V + Inhibitor
2 max
1
Even at high V
2 max,app
substrate levels,
inhibitor binds,
[E]t < [ES] Km,app Km
.
[Substrate]
Vmax,app < Vmax
81
Effect of Temperature
• Enzymes have an optimum temperature at
which they are most active
• The optimum temperature for most
human enzymes is normal body
temperature, 37 oC
• Above optimum temperature, enzymes
lose activity due to disruption of
intermolecular forces stabilizing the
tertiary structure.
82
0°C
• Low temperatures - low Kinetic
Energy of enzymes and substrates.
• No/Very few enzyme-substrate

complexes are formed.
• Enzymes are inactivated.

83
20°C (increasing temperature)
• Increasing the temperature will lead to the
increase in kinetic energy of enzyme and
substrate molecules.
• Enzyme and substrate molecules move with
increasing speed and collide more frequently
with each other.
• This increases the rate of enzyme-substrate
complex formation
• This increases the rate of enzyme-substrate
complex formation and product formation.
84
37°C
• As the temperature continues to increase,
the rate of enzyme activity also increases
until the optimal temperature is reached.
• Optimal temperature is the temperature

at which the enzyme works best. Rate of
product formation is highest!

85
Beyond Optimal Temperatures
• At high temperatures (>60°C), weak
bonds within the enzyme molecule are
broken
• Enzyme loses its shape and its active site.
• Loss of shape leads to a loss of function.
Enzyme is said to have denatured
• Denaturation is the change in 3D
structure of an enzyme or any other
protein caused by heat or chemicals such
as acids or alkali, causing it to lose its
function.
86
Denaturation
Different enzymes denature at different temperatures.

Most enzymes denature at temperatures higher than
60°C. However, there are some enzymes that stay
active even at high temperatures like 80°C (Enzymes
in the bacteria Thermus aquaticus)
87
Effect of PH
• When the enzyme environment is changed
by pH, its tertiary structure is disrupted,
altering the active site and causing the
enzyme‟s activity to decrease.
• Enzymes are most active at a pH known as

their optimum pH.
• At optimum pH, the enzyme maintains its

tertiary structure and its active site.
88

02.enzyme Kinetics

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02.enzyme Kinetics

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Adama Science &

02. Enzyme Kinetics

February 26, 2021 Slide By Eba A

Alpha amylase starch Glucose. Maltose and

Lipase Fat Fatty acid, glycerol

maltase maltose glucose

urease Urea Ammonia , Carbondioxide

cellobiase cellobiose glucose

February 26, 2021 Slide By Eba A

Alcohol Ethanol + NAD+ Acetaldehyde +

Glucose Oxidase D-glucose + Gluconic acid

HFCS is used in soft drink production

 The change of product and  The effect of substrate

 If we measure the initial reaction rate at different levels of

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

• The concentration of the enzyme-substrate

February 26, 2021 Slide By Eba A

• Substituting [E] in the above equation with enzyme

February 26, 2021 Slide By Eba A

ES remains constant while S is converted to P. Here

Using [S] to represent [ES] yields

February 26, 2021 Slide By Eba A

Where Vm  k [ E 0 ] same as that for rapid equilibrium assumption.

February 26, 2021 Slide By Eba A

Equation: Vm [S] Vm [S]

February 26, 2021 Slide By Eba A

• Derive the rate equation by employing

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

• The slope is –Km

Langmuir plot ([S]/V

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

Km, not the Vmax

+ Inhibitor Vmax,app = Vmax

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

Remains that of Michaelis-menten equation

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

• Since noncompetitive inhibitors do not interfere

February 26, 2021 Slide By Eba A

Vmax,app < Vmax

February 26, 2021 Slide By Eba A

EI + S ESI of the enzyme for the

I I Menten equation, the v vs.

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

February 26, 2021 Slide By Eba A

Remains in Michaelis-menten equation

February 26, 2021 Slide By Eba A