Kinetic parameters for several

enzymes
It is important to have a feel for the magnitudes of the
kinetic constants, k and 1/KM, for certain enzymes. The
table in the next slide shows the range of values
encountered for several different enzymes. Notice that
almost all the experiments reported were performed at
moderate temperatures and pH values. The exception
is pepsin, which has the task of hydrolyzing proteins in
the acid environment of the stomach. Consequently,
the enzyme has the greatest activity under the acidic
conditions employed in the experimental
determination of its kinetic parameters.

Michaelis-Menten Constants for Some Enzyme-Substrate Systems

Enzyme

Source

Substrate

KM (mM)

Alcohol dehydrogenase

Saccharomyces cerevisiase

Ethanol

13.0

-Amylase

Bacillus stearothermophilus
Porcine pancreas

Starch
Starch

1.0
0.4

-Amylase

Sweet potato

Amylose

0.07

Aspartase

Bacillus cadaveris

L-Aspartate

30.0

-Galactosidase

Escherichia coli

Lactose

3.85

Glucose oxidase

Aspergillus niger
Penicillium notatum

D-Glucose
D-Glucose

33.0
9.6

Histidase

Pseudomonas fluorescens

L-Histidine

8.9

Invertase

Saccharomyces cerevisiae
Neurospora crassa

Sucrose
Sucrose

9.1
6.1

Lactate dehydrogenase

Bacillus subtilis

Lactate

30.0

Penicillinase

Bacillus licheniformis

Benzylpenicillin

0.049

Urease

Jack bean

Urea

10.5

Evaluation of Michaelis-Menten
Parameters
A series of batch runs with different levels of
substrate concentration is made in order to
estimate the values of the kinetic parameters.
The results are plotted graphically so that the
validity of the kinetic model can be tested and
the values of the kinetic parameters can be
estimated.

The most straightforward way is to plot r

against CS

The asymptote for r will be rmax and KM is

equal to CS when r = 0.5rmax. However, this
is an unsatisfactory plot in estimating rmax
and KM because it is difficult to test the
validity of the kinetic model. Therefore,
the Michaelis-Menten equation is usually
rearranged so that the results can be
plotted as a straight line.

The Michaelis-Menten equation is

rearranged to be expressed in linear form.
Some of the better known methods are:
(a) Langmuir plot
(b) Lineweaver-Burk plot
(c) Eadie-Hofstee plot

LANGMUIR PLOT
Also known as Hanes-Woolf plot
Proponents of this method are:
Charles Samuel Hanes
Barnet Woolf

This equation was given by Langmuir for the

treatment of data from the adsorption of gas
on a solid surface.

CS K M
1

CS
r
rmax rmax
Refer to Figure 2.4, p. 24, James Lee

Slope: 1/rmax and intercept: KM/rmax

LINEWEAVER-BURK PLOT
Also known as double reciprocal plot
Proponents are:
Hans Lineweaver
Dean Burk

KM 1
1
1

r rmax rmax CS
Refer to Figure 2.5, p. 25, James Lee
Slope: KM/rmax
Intercept: 1/rmax

EADIE-HOFSTEE DIAGRAM
Also known as:
Woolf-Eadie-Augustinsson-Hofstee
Eadie-Augustinsson

r rmax K M

r
CS

Refer to Figure 2.6, p. 25, James Lee

Slope: KM
Intercept: rmax

Observations:
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. However, 1/r
approaches infinity as CS decreases, which gives
undue weight to inaccurate measurements made at
low substrate concentrations, and insufficient weight
to the more accurate measurements at high
substrate concentrations. The points on the line in
Figure 2.5 represent seven equally spaced substrate
concentrations. The space between the points
increases with the decrease in CS.

Observations
The Eadie-Hofstee plot gives slightly better
weighting of the data than the LineweaverBurk plot. A disadvantage of this plot is that
the rate of reaction r appears in both
coordinates while it is usually regarded as a
dependent variable.
Based on the data distribution, the Langmuir
plot is the most satisfactory of the three, since
the points are equally spaced.

Summary:
In conclusion, the values of MM kinetic parameters,
rmax and KM can be estimated as follows:

1. 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
with respect to time.
2. Estimate the initial rate of reaction from the
CS or CP versus time curves for different
initial substrate concentrations.

Summary:
In conclusion, the values of MM kinetic parameters,
rmax and KM can be estimated as follows:

3. Estimate the kinetic parameters by plotting

one of the three plots. It is important to
examine the data points so that you may not
include the points which deviate
systematically from the kinetic model.

Example 2.3
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.
CS
(mmol/L)

10

15

20

Initial rxn
rate, r
(mmol/L
min)

0.20

0.22

0.30

0.45

0.41

0.50

0.40

0.33

Example 2.3
a. Evaluate the Michaelis-Menten kinetic
parameters by employing the Langmuir plot, the
Lineweaver-Burk plot, the Eadie-Hofstee plot. In
evaluating the kinetic parameters, do not include
data points which deviate systematically from
the Michaelis-Menten model and explain the
reason for deviation.
b. Which of the three methods employed gives the
best estimate of the kinetic parameters?
c. Repeat part (a) by using all data.

Example 2.3
a.

Parameter

Langmuir

LineweaverBurk

Eadie-Hofstee

4.641709208

1.945013423

0.5386020591

1.586604846

3.457511152

1.892320771

0.974502384

0.919919746

0.818534391

rmax,
mmol/Lmin

0.6303

0.5141

0.5386

KM, mmol/L

2.9256

1.7776

1.8923

b. Langmuir plot

Example 2.3
c.

Parameter

Langmuir

LineweaverBurk

Eadie-Hofstee

0.102919802

2.224903446

0.4499423397

2.717533491

3.038672816

1.21143558

0.9727197005 0.8846686122 0.660136829

rmax,
mmol/Lmin

0.3680

0.4495

0.4499

KM, mmol/L

0.0379

1.3658

1.2114

Problem 2.4
Eadie (1942) measured the initial reaction rate
of hydrolysis of acetylcholine (substrate) by
dog serum (source of enzyme) and obtained
the following data:
Substrate
Concn (mol/L)
Initial Rxn
Rate
(mmol/Lmin)

0.0032

0.0049

0.0062

0.0080

0.0095

0.111

0.148

0.143

0.166

0.200

Problem 2.4
Evaluate the Michaelis-Menten kinetic
parameters employing (a) the Langmuir plot,
(b) the Lineweaver-Burk plot, and (c) the
Eadie-Hofstee plot.

Problem 2.4
(a) By Langmuir plot:
A = 0.01912464593
B = 3.313305715
R = 0.9400618089
(a) By Lineweaver-Burk plot:
A = 3.63421308
B = 0.0171910583
R = 0.9563479914
(a) By Eadie-Hofstee plot:
A = 0.2644954792
B = 4.27314138210 3
R = 0.8114158691

rmax = 0.3018 mol/Lmin

KM = 5.771810 3 mol/L

rmax = 0.2752 mol/Lmin

KM = 4.730310 3 mol/L

rmax = 0.2645 mol/Lmin

KM = 4.273110 3 mol/L

Problem 2.14
Eadie (1942) measured the initial reaction rate
of hydrolysis of acetylcholine (substrate) by
dog serum (source of enzyme) in the absence
and presence of prostigmine (inhibitor),
1.510 7 mol/L and obtained the following
data:

Problem 2.14
CS mol/L

r, mol/Lmin
Absence of Prostigmine

Presence of Prostigmine

0.0032

0.111

0.059

0.0049

0.148

0.071

0.0062

0.143

0.091

0.0080

0.166

0.111

0.0095

0.200

0.125

a. Evaluate the Michaelis-Menten kinetic parameters in the

presence and absence of prostigmine by employing:
i. Langmuir plot
ii. Lineweaver-Burk plot
iii. Eadie-Hofstee plot
b. Is prostigmine competitive or noncompetitive?

Problem 2.14
Given:
CS mol/L

r, mol/Lmin
Absence of Prostigmine

Presence of Prostigmine

0.0032

0.111

0.059

0.0049

0.148

0.071

0.0062

0.143

0.091

0.0080

0.166

0.111

0.0095

0.200

0.125

Problem 2.14
Required:
a. MM kinetic parameters (KM and rmax) by
employing
a.1 Langmuir plot
a.2 Lineweaver-Burk plot
a.3 Eadie-Hofstee plot
b. Is prostigmine competitive or noncompetitive
inhibitor?

Problem 2.14
Solution:
a.1 Langmuir Plot
CS K M
1

CS
r
rmax rmax

w/o prostigmine:
rmax = 0.3018
KM = 5.772110 3
R = 0.9400618089

w/ prostigmine
rmax = 0.3346
KM = 0.0164
R = 0.9020508553

Problem 2.14
0.09

0.08

y = 2.9883x + 0.0489

0.07

0.06

y = 3.3133x + 0.0191

0.05

w/o inhibition
w/ inhibition
Linear (w/o inhibition)

0.04

Linear (w/ inhibition)

0.03

0.02

0.01

0
0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.01

Problem 2.14
Solution:
a.2 Lineweaver-Burk Plot
KM 1
1
1

r rmax rmax CS

w/o prostigmine:
rmax = 0.2752
KM = 4.730310 3
R = 0.9563479914

w/ prostigmine
rmax = 0.2613
KM = 0.0115
R = 0.9783254623

Problem 2.14
20
18

y = 0.0439x + 3.8266

16
14
12

w/o inhibitor
w/ inhibitor

10

y = 0.0172x + 3.6342

Linear (w/o inhibitor)

Linear (w/ inhibitor)

8
6
4
2
0
0

50

100

150

200

250

300

350

Problem 2.14
Solution:
a.3 Eadie-Hofstee Plot
r rmax K M

w/o prostigmine:
rmax = 0.2645
KM = 4.273110 3
R = 0.8114158691

r
CS

w/ prostigmine
rmax = 0.2555
KM = 0.0110
R = 0.8256238796

Problem 2.14
0.25

0.2

0.15

w/o inhibitor
w/ inhibitor
Linear (w/o inhibitor)

y = -0.0043x + 0.2645

0.1

Linear (w/ inhibitor)

y = -0.011x + 0.2555

0.05

0
0

10

15

20

25

30

35

40

Problem 2.14
Solution:
Summary
Langmuir

Lineweaver-Burk

Eadie-Hofstee

w/o I

w/ I

w/o I

w/ I

w/o I

w/ I

rmax,
mol/Lmin

0.3018

0.3346

0.2752

0.2613

0.2645

0.2555

KM, mol/L

5.772110 3

0.0164

4.730310 3

0.0115

4.273110 3

0.0110

0.9401

0.9021

0.9563

0.9783

0.8114

0.8256

b. Since maximum reaction rate did not change

greatly relative to KM, the inhibitor is
COMPETITIVE.

Problem 2.17
The initial rate of reaction for the enzymatic
cleavage of deoxyguanosine triphosphate was
measured as a function of initial substrate
concentration as follows (Kornberg et al., J.
Biol. Chem., 233, 159, 1958):
CS (mol/L)
6.7
3.5
1.7

r (mol/Lmin)
0.30
0.25
0.16

Problem 2.17
a. Calculate the Michaelis-Menten constants of
the above equation by:
i. Langmuir plot
ii. Lineweaver-Burk plot
iii. Eadie-Hofstee plot

Problem 2.17
b. When the inhibitor was added, the initial reaction
rate was decreased as follows:
CS (mol/L)
6.7
3.5
1.7

Inhibitor
146
146
146

r (mol/Lmin)
0.11
0.08
0.06

Is this competitive or noncompetitive inhibition? Justify

your answer by showing the effect of the inhibitor
graphically employing the three plots. [Contributed
by Professor Gary F. Bennett, The University of
Toledo, Toledo, OH]

Problem 2.17
Given:
CS (mol/L)
6.7
3.5
1.7

CS (mol/L)
6.7
3.5
1.7

r (mol/Lmin)
0.30
0.25
0.16

Inhibitor
146
146
146

r (mol/Lmin)
0.11
0.08
0.06

Problem 2.17
Required:
a. MM kinetic parameters (rmax and KM) with
and without the presence of the inhibitor by
employing:
i. Langmuir plot
ii. Lineweaver-Burk plot
iii. Eadie-Hofstee plot
b. Is the inhibition competitive or
noncompetitive?

Problem 2.17
Solution:
a.1 Langmuir Plot
CS K M
1

CS
r
rmax rmax

w/o inhibitor:
rmax = 0.4215
KM = 2.6317
R = 0.9968395842

w/ inhibitor:
rmax = 0.1567
KM = 2.9807
R = 0.9916395194

Problem 2.17
70

y = 6.3809x + 19.02

60

50

40

w/o inhibitor

w/ inhibitor
Linear (w/o inhibitor)

30

Linear (w/ inhibitor)

y = 2.3722x + 6.2429

20

10

0
0

Problem 2.17
Solution:
a.2. Lineweaver-Burk plot
KM 1
1
1

r rmax rmax CS

w/o inhibitor:
rmax = 0.4511
KM = 3.0566
R = 0.9960691972

w/ inhibitor:
rmax = 0.1415
KM = 2.3613
R = 0.9876450915

Problem 2.17
18

y = 16.68x + 7.0637
16

14

12

w/o inhibitor

10

w/ inhibitor
Linear (w/o inhibitor)

Linear (w/ inhibitor)

y = 6.7758x + 2.2168

0
0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Problem 2.17
Solution:
a.3. Eadie-Hofstee
r rmax K M

w/o inhibitor:
rmax = 0.4336
KM = 2.8096
R = 0.9780668301

r
CS

w/ inhibitor:
rmax = 0.1457
KM = 2.5083
R = 0.9563966776

Problem 2.17
0.35

0.3

0.25

0.2

w/o inhibitor
w/ inhibitor

y = -2.8096x + 0.4336

Linear (w/o inhibitor)

0.15

Linear (w/ inhibitor)

0.1

y = -2.5083x + 0.1457

0.05

0
0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

Problem 2.17
Solution:
Summary
Langmuir

Lineweaver-Burk

Eadie-Hofstee

MM kinetic
parameters

w/o I

w/ I

w/o I

w/ I

w/o I

w/ I

rmax, mol/Lmin

0.4215

0.1567

0.4511

0.1415

0.4336

0.1457

KM, mol/L

2.6317

2.9807

3.0566

2.3613

2.8096

2.5083

0.9968

0.9916

0.9961

0.9263

0.9781

0.9564

b. Since KM did not change greatly relative to rmax, then

the inhibition is NONCOMPETITIVE.

SEATWORK Problem 2.18

The enzyme, cathepsin, hydrolyzes L-glutamyl-Ltyrosine to carbobenzoxy-L-glutamic acid and Ltyrosine. It has been found (Frantz and Stephenson,
J. Biol. Chem., 169, 359, 1947) that the glutamic acid
formed in the hydrolysis, inhibits (competitively) the
progress of the reaction by forming a complex with
cathepsin. The course of the reaction is followed by
adding tyrosine decarboxylase which evolves CO2.

SEATWORK Problem 2.18

Substrate, mol/mL

Inhibitor, mol/mL

Rate of CO2 Generation,

mol/mLmin

4.7

0.0434

4.7

7.57

0.0285

4.7

30.30

0.0133

10.8

0.0713

10.8

7.58

0.0512

10.8

30.30

0.0266

30.3

0.1111

30.3

7.58

0.0909

30.3

30.30

0.0581

SEATWORK Problem 2.18

Calculate (a) the value of Michaelis-Menten
constants of the enzyme, KS, and (b) the
dissociation constant of enzyme-inhibitor
complex, KI. [Contributed by Professor Gary F.
Bennett, The University of Toledo, Toledo, OH]

Enzyme Reactor and Simple Kinetics

Bioreactor device within which biochemical
transformations are caused by the action of
enzymes or living cells
Fermenter bioreactor in which the transformation
is carried out by living cells or in vivo cellular
components (that is, enzymes)
Enzyme reactor bioreactor employing enzymes;
the term is used to distinguish it from the
bioreactor in which employs living cells

Batch / Plug-Flow Reactor (SteadyState)

The simplest reactor configuration for any
enzyme reaction is the batch mode. A batch
enzyme reactor is normally equipped with an
agitator to mix the reactant, and the pH of the
reactant is maintained by employing 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.

Schematic Diagram of a Batch Reactor

MM General Rate Equation

dCS
rmax CS

dt
K M CS
Rearranging, and integrating yields
CS

CS 0

K CS
M
CS

dCS rmax dt

Integrated Form:

C S0

CS0 CS rmax t
CS
The eqn above shows how CS is changing with
respect to time. With known values of rmax and
KM, the change in CS with time in a batch
reactor can be predicted from this eqn.
K M ln

Plug-flow enzyme reactor (tubular-flow enzyme reactor)

In a plug-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 long tube and lack of stirring device
prevents complete mixing of the fluid in the tube.
Therefore, the properties of the flowing stream will vary
in both longitudinal and radial directions. Since the
variation in radial direction is small compared to that in
the longitudinal direction, it is called a plug-flow reactor.
If a plug-flow reactor is operated at steady-state, the
properties will be constant with respect to time. The
ideal plug-flow enzyme reactor can approximate the
long tube, packed-bed, and hollow fiber, or multistaged
reactor.

Schematic Diagram of Plug-Flow Enzyme Reactor

Linear Form of Prior Given Eqn:

C S0 C S

C S0
ln
CS

K M rmax

t
C S0
ln
CS

Continuous Stirred-Tank Reactor

A continuous stirred-tank
reactor (CSTR) is an ideal
reactor w/c is based on the
assumption that the reactor
contents are well mixed.
Therefore, the concentrations of the various
components of the outlet stream are assumed to be
the same as the concentrations of these components in
the reactor.

Continuous Stirred-Tank Reactor

Substrate balance in CSTR:
Input Output Consumption = Accumulation
FC S0

dC S
FC S rS V V
dt

where:
F = flow rate
V = volume
rS = rate of substrate consumption
dCS/dt = change of substrate concn
For a batch reactor: F = 0 and rS = dCS/dt

Continuous Stirred-Tank Reactor

For a steady-state CSTR, dCS/dt = 0, and rS is given by
Michaelis-Menten rate equation as:
rmax C S
F
1
D
V
C S 0 C S K M C S

where:
D = dilution ratio
Rearranging the equation above gives the linear
relationship
C S K M rmax

C S
C S0 C S

Problem 2.6
A carbohydrate (S) decomposes in the presence of an
enzyme (E). The Michaelis-Menten kinetic parameters
were found as follows:
KM = 200 mol/m3
rmax = 100 mol/m3min
a. Prepare a CS versus t curve when the initial substrate
concentration is 300 mol/m3.
b. Assume that you obtained the CS versus t curve you
calculated in part (a) experimentally. Estimate KM and rmax
by plotting the (CS0 CS)/ln(CS0/CS) versus t/ln(CS0/CS)
curve.

Problem 2.6
c. Chemostat (continuously stirred-tank reactor) runs with
various flow rates were carried out. If the inlet substrate
concentration is 300 mol/m3 and the flow rate is 100
cm3/min, what is the steady-state substrate
concentration of the outlet? The reactor volume is 300
cm3. Assume that the enzyme concentration in the
reactor is constant so that the same kinetic parameters
can be used.

Problem 2.6
Given:

KM = 200 mol/m3
rmax = 100 mol/m3min
CS0 = 300 mol/m3

Problem 2.6

Required:
a. CS vs t graph
b. KM and rmax suppose values obtained in (a)
were determined experimentally.
c. CSTR configuration, find CS outlet

CS0 = 300 mol/m3

F = 100 cm3/min
V = 300 cm3

Problem 2.6

Solution
a. Table
K M ln

C S0
CS

C S0 C S rmax t

CS, mol/m3

t, min

250
200

0.8646
1.8109

150
100

2.8863
4.1972

50
0

6.0835
---------

Problem 2.6

Solution
b. Table for Linear Graph:
C S0 C S
C S0
ln
CS

K M rmax

t
C S0
ln
CS

By LR:
KM = 200.0639 mol/m3
rmax = 100.0165 mol/m3min

CS, mol/m3

t, min

t/ln(CS0/CS)

(CS0 CS)/ln(CS0/CS)

250

0.8646

4.7422

274.2407

200

1.8109

4.4662

246.6303

150

2.8863

4.1641

216.4043

100

4.1972

3.8205

182.0478

50

6.0835

3.3953

139.5277

----------

----------

----------

Problem 2.6

Solution
c. Continuous stirred tank reactor (CSTR)
By applying the derived equation:
C S K M rmax

C S
C S0 C S

CS = 164.5751 mol/m3
Other Root = 364.6895

Problem 2.7
The KM value of an enzyme is known to be 0.01
mol/L. To measure the maximum reaction rate
catalyzed by the enzyme, you measured the
initial rate of reaction and found that 10
percent of the initial substrate concentration
was consumed in 5 minutes. The initial
substrate concentration is 3.410 4 mol/L.
Assume that the reaction can be expressed by
the Michaelis-Menten kinetics.

Problem 2.7
a. What is the maximum reaction rate?
b. What is the concentration of substrate after
15 minutes
For a BATCH enzyme reactor:
rmax = 2.1752104 mol/Lmin
CS = 2.4762104 mol/L

Problem 2.8
A substrate is converted to a product by the catalytic
action of an enzyme. Assume the MichaelisMenten kinetic parameters for this enzyme
reaction are:
KM = 0.03 mol/L
rmax = 13 mol/Lmin
a. What should be the size of a steady-state CSTR to
convert 95 percent of the incoming substrate (CS0 =
10 mol/L) with a flow rate of 10 L/h.
b. What should be the size of the reactor if you
employ a plug-flow reactor instead of the CSTR in
(a)?

Problem 2.8
Given
KM = 0.03 mol/L
rmax = 13 mol/Lmin
F = 10 L/h
CS0 = 10 mol/L

CS = ?

95% conversion

Problem 2.8
Required
a. V for 95% conversion
b. V if plug-flow reactor is employed

VCSTR = 0.1291 L
VPFR = 0.1229 L

Problem 2.9
A substrate is decomposed in the presence of an enzyme
according to the Michaelis-Menten equation with the
following kinetic parameters:
KM = 10 g/L
rmax = 7 g/Lmin
If we operate two one-liter CSTRs in series at steady-state,
what will be the concentration of substrate leaving the
second reactor? The flow rate is 0.5 L/min. The inlet
substrate concentration is 50 g/L and the enzyme
concentration in the two reactors is maintained at the
same value all of the time. Is the two-reactor system more
efficient than one reactor whose volume is equal to the
sum of the two reactors?

Problem 2.9
1 = 38.8650 g/L
1 = 12.8650
2 = 28.5012 g/L
2 = 13.6362

%Conversion = 42.9976%
= 29.1517 g/L
= 17.1517
%Conversion = 41.6966%

Since, % conversion of two CSTRs in series is higher, then it is the better configuration.

Problem 2.1
In order to measure the enzyme activity and the
initial rate of reaction, 5 mL of cellobiose (100
mol/mL) and 44 mL of buffer solution were
placed in a stirred vessel. The reaction was
initiated by adding 1 mL of enzyme (glucosidase) solution which contained 0.1 mg
of protein per mL. At 1, 5, 10, 15, and 30
minutes, 0.1 mL of sample was removed from
reaction mixture and its glucose content was
measured. The result were as follows:

Problem 2.1
Time, min

Glucose Concentration, mol/mL)

0.05

0.23

10

0.38

15

0.52

30

1.03

a. What is the activity of the -glucosidase in units/mL of

enzyme solution and in units/mg protein? A unit is defined as
the enzyme activity which can produce 1 mol of product per
minute?
b. What is the initial rate of reaction?

Other Influences on Enzyme Activity

Some chemical and physical conditions affect
the rate of an enzyme reaction. Some of these
factors are the concentration of various
components (substrate, product, enzyme,
cofactor, and so on), pH, temperature, and
shear.

Effect of pH
The pH of the solution strongly influences the
rate of enzyme reaction both in vivo and in
vitro. The optimum pH is different for each
enzyme.
Examples:
Pepsin (from stomach) 2 < pH < 3.3
Amylase (from saliva) pH = 6.8
Chymotripsin (from pancreas) 7 < pH < 8

Effect of pH

The typical relationship b/n the rxn velocity and pH shows a

bell-shaped curve.

Effect of pH
Reasons 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

Effect of pH
2. The amino acid residues possess basic,
neutral, or acid side groups which can be
positively or negatively charged at any given
pH.

Glutamic acid is acidic at lower pH. As the pH is

increased, glutamic acid is ionized.

Effect of pH
Ionization is expressed according to:

In eqbm,

When C A C A , pH is equal to pK. For glutamic

acid, pK = 4.5.

Effect of pH
Lysine is basic in the range of higher pH value.
As the pH is decreased, lysine is ionized as

pK value of lysine is 10.0 at which half of the

residues are ionized.

Effect of pH
3. An enzyme is catalytically active when the
amino acid residues at the active site each
possess a particular charge. Therefore, the
fraction of the catalytically active enzyme
depends on the pH.

Effect of pH
Conclusion
Suppose that one residue of each of these two
amino acids, glutamic acid and lysine, is
present at the active site of an enzyme
molecule and that, for example, the charged
form of both amino acids must be present if
that enzyme is to function. Since glutamic acid
is charged when its pH 4.5 and lysine is
charged when its pH 10.0, the enzyme will
be most active when 4.5 pH 10.0 as shown
in the given figure.

Effect of Temperature
The rate of enzyme-catalyzed reactions
increases with temperature up to a certain
limit. Above a certain temperature, enzyme
activity decreases with temperature because
of enzyme denaturation.

Effect of Temperature
Figure below depicts the variation of reaction
rate with temperature and the presence of an
optimal temperature.

Effect of Temperature
Temperature activation: ascending part; the
rate varies according to the Arrhenius
equation in this region.

r k 2C E
where

k2 Ae

Ea / RT

Effect of Temperature
where
Ea = activation energy
CE = active enzyme concentration
Plot of ln(r) versus 1/T results in a line with slope
Ea/R

Effect of Temperature
Temperature deactivation/thermal denaturation
dC E

kd CE
dt

CE CE0 e kd t

Denaturation constant, kd, varies with

temperature according to the Arrhenius eqn
kd Ad e Ea / RT

Effect of Temperature
Consequently
r Ae Ea / RT CE0 e kd t

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.

Effect of Shear
Charm and Wong (1970) showed that the enzymes
catalase, rennet, and carboxypeptidase were partially
inactivated when subjected to shear in a coaxial
cylinder viscometer. The remaining activity could be
correlated with a dimensionless group . In the case
of catalase, about 50 percent of the activity was lost
when was 0.5107.
= shear rate
= time of exposure to shear

Effect of Shear
Thomas and Dunnill (1979) studied the effect of shear
on catalase and urease activities by using a coaxial
cylindrical viscometer that was sealed to prevent any
air-liquid contact. They found that there was no
significant loss of enzyme activity due to shear force
alone at shear rates up to 106 sec1. They reasoned
that the deactivation observed by Charm and Wong
(1970) was the result of combination of shear, airliquid interface, and some other effects which are
not fully understood. Charm and Wong did not seal
their shear apparatus.

Effect of Shear
Recently, this as further confirmed, as cellulase
deactivation due to the interfacial effect combined
with the shear effect was found to be far more
severe and extensive than that due to the shear
effect alone (Jones and Lee, 1988).

Objective Questions / Problems

1. T/F? The Ea calculated from the Arrhenius equation
gives an exact value.
Ans.: F. Ea is an average or apparent value.
2. Describe the relationship between temperature and
kd and give examples.
Ans.: As the temperature increases, the rate constant
decreases when the equation is plotted. The same
is true when the temperature decreases, the rate
constant increases. From this connection, the rate
constant is inversely proportional to temperature.

Objective Questions / Problems

3. Using the following information:
A = 11014 sec 1
Ea = 75103 J/mol
R = 8.314 J/molK
Calculate k at 27C with proper units.
4. Using the information in problem 3, calculate k at
37C with proper units.
5. Calculate the value of Ea given:
kd1 = 7.78107 at T1 = 273 K
kd2 = 3.46105 at T2 = 298 K

Answers
kd = 8.8592/sec
kd = 23.3476/sec
Ea = 102,670.8716 J/mol