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Kinetic parameters for several

enzymes
It is important to have a feel for the magnitudes of
the kinetic constants, k and 1/K M, 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.

Kinetic parameters for several


enzymes
Enzyme
Pepsin

Trypsin

Chymotryp
sin

Substrate

Temp,
C

pH

k3 , s 1

1/KM, M
1

Carbobenzoxy-Ltyrosine ethyl ether

31.6

4.0 0.00108

530

Carbobenzoxy-Lglutamyl-L-tyrosine

31.6

4.0 0.00141

560

Benzoyl-Largininamide

25.5

7.8

27.0

480

Chymotrypsinogen

19.6

7.5

2,900

<770

Saturin

24.5

7.5

13,100

400

Benzoyl-L-arginine
ester

25.0

8.0

26.7

12,500

Methyl
hydrocinnamate

25.0

7.8

0.026

256

Methyl dl--chloro-phenylpropionate

25.0

7.8

0.135

83.3

Kinetic parameters for several


enzymes
Enzyme

Substrate
Methyl l-phenyllactate

Temp,
C

pH

25.0

7.8

k3 , s 1

1/KM, M
1

1.38

100

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 C S
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.

Observations
The Eadie-Hofstee plot gives slightly
better weighting of the data than the
Lineweaver-Burk 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
explained in this section. 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

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.

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)

0.0032

0.0049

0.0062

0.0080

0.0095

Initial Rxn
Rate
(mmol/Lmi
n)

0.111

0.148

0.143

0.166

0.200

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

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: w/ prostigmine


rmax = 0.3018
rmax = 0.3346
KM = 5.772110 3 KM = 0.0164
R = 0.9401

R = 0.9021

Problem 2.14
0.08
f(x) = 2.99x + 0.05
0.07

0.06

0.05

f(x) = 3.31x + 0.02


w/o inhibition
Linear (w/o inhibition)

0.04

w/ inhibition
Linear (w/ inhibition)
0.03

0.02

0.01

0
0

0.01

0.01

0.01

0.01

0.01

0.01

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

r rmax rmax C S

w/o prostigmine: w/ prostigmine


rmax = 0.2752
rmax = 0.2613
KM = 4.730310 3 KM = 0.0115
R = 0.9563
R = 0.9783

Problem 2.14
18
f(x) = 0.04x + 3.83
16

14

12

10

w/o inhibitor
Linear (w/o inhibitor)

f(x) = 0.02x + 3.63

w/ inhibitor
Linear (w/ inhibitor)

0
50

100

150

200

250

300

350

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

r
CS

w/o prostigmine: w/ prostigmine


rmax = 0.2645
rmax = 0.2555
KM = 4.273110 3 KM = 0.0110
R = 0.8114
R = 0.8256

Problem 2.14
0.25

0.2

f(x) = - 0x + 0.26
0.15
w/o inhibitor
Linear (w/o inhibitor)
w/ inhibitor
f(x) = - 0.01x + 0.26

0.1

Linear (w/ inhibitor)

0.05

0
10

15

20

25

30

35

40

Problem 2.14
Solution:
Summary
Langmuir

Lineweaver-Burk

Eadie-Hofstee

rmax,
mol/Lmi
n

0.3018

0.3346

0.2752

0.2613

0.2645

0.2555

KM,
mol/L

5.7721
10 3

0.0164

4.7303
10 3

0.0115

4.2731
10 3

0.0110

0.9401
0.9021
0.9563
0.9783
0.8114
b. Since
maximum
reaction
rate
did 0.8256
not change greatly relative to KM, the
inhibitor is COMPETITIVE.

Problem 2.14
Solution:
Summary
Langmuir
MM
w/o
kinetic
prostigmi
paramete
ne
rs

Lineweaver-Burk

Eadie-Hofstee

w/
prostigmi
ne

w/o
prostigmi
ne

w/
prostigmi
ne

w/o
prostigmi
ne

w/
prostigmi
ne

rmax,
mol/Lmin

0.3018

0.3346

0.2752

0.2613

0.2645

0.2555

KM, mol/L

5.77211
03

0.0164

4.73031
03

0.0115

4.27311
03

0.0110

b. Since
maximum
reaction
rate 0.9783
did not change
0.9401
0.9021
0.9563
0.8114
greatly relative to KM, the inhibitor is
COMPETITIVE.

0.8256

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,
CS (mol/L)
r (mol/Lmin)
159, 1958):
6.7
3.5
1.7

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)

Inhibitor

r
(mol/Lmi
n)
6.7
146
0.11
3.5
146
0.08
1.7
0.06
Is this competitive
or146
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)

r (mol/Lmin)

6.7
3.5
1.7

0.30
0.25
0.16

CS
(mol/L)

Inhibitor

6.7
3.5
1.7

146
146
146

r
(mol/Lmi
n)
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:
w/ inhibitor:
rmax = 0.4215
rmax = 0.1567
KM = 2.6317 KM = 2.9807
R = 0.9968
R = 0.9916

Problem 2.17
70

60

f(x) = 6.38x + 19.02

50

40

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

30

Linear (w/ inhibitor)

f(x) = 2.37x + 6.24

20

10

0
1

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

r rmax rmax C S

w/o inhibitor:
w/ inhibitor:
rmax = 0.4511
rmax = 0.1415
KM = 3.0566 KM = 2.3613
R = 0.9961
R = 0.9876

Problem 2.17
18

16

f(x) = 16.68x + 7.06

14

12

10

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

Linear (w/ inhibitor)


f(x) = 6.78x + 2.22

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

r
CS

w/o inhibitor:
w/ inhibitor:
rmax = 0.4336
rmax = 0.1457
KM = 2.8096 KM = 2.5083
R = 0.9781
R = 0.9564

Problem 2.17
0.35

0.3

f(x) = - 2.81x + 0.43

0.25

0.2

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

0.15

0.1

Linear (w/ inhibitor)

f(x) = - 2.51x + 0.15

0.05

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
paramete
rs

w/o
inhibitor

w/
inhibitor

w/o
inhibitor

w/
inhibitor

w/o
inhibitor

w/
inhibitor

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 Lglutamyl-L-tyrosine to carbobenzoxy-Lglutamic acid and L-tyrosine. 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 MichaelisMenten 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]

SEATWORK Problem 2.18


Given:
CS, mol/mL

CI, mol/mL

Rate of CO2
Generation, r,
mol/mLmin

4.7

0.0434

10.8

0.0713

30.3

0.1111

4.7

7.57

0.0285

10.8

7.58

0.0512

30.3

7.58

0.0909

4.7

30.30

0.0133

10.8

30.30

0.0266

30.3

30.30

0.0581

SEATWORK Problem 2.18


Required:
a. MM constant of the enzyme, KS
b. Dissociation constant of enzymeinhibitor complex, KI

SEATWORK Problem 2.18


Solution:
Any method can be used to solve for
KM and rmax
*By Langmuir Plot
CS K M
1

CS
r
rmax r max

@CI = 0
rmax = 0.1569 mol/mLmin R = 0.9997
KM = 12.5840 mol/mL

SEATWORK Problem 2.18


Solution:
@ CI = 7.58 mol/mL
rmax = 0.1537 mol/mLmin
0.9993
KM C
=I =
21.0719
@
30.30 mol/mL
rmax = 0.1560 mol/mLmin
0.9967
KM = 51.3369 mol/mL

R=

R=

SEATWORK Problem 2.18


Solution:
It is observed from the values of KM
and rmax that the inhibition is

CI

is given
K MI K S 1KMI

COMPETITIVE. Hence,
KI

by:

Making use of the values solved for K MI:


KMI = 21.0719 mol/mL @ CI = 7.58 mol/mL
KMI = 51.3369 mol/mL @ CI = 30.30 mol/mL

SEATWORK Problem 2.18


Solution:
Making use of the values solved for
KMI:
KMI = 21.0719 mol/mL @ CI = 7.58
mol/mL
We
now have two equations and two
KMIunknowns
= 51.3369
mol/mL
@ CI = 30.30

given
by:
7.58

21.0719 K S 1
KI
mol/mL

30.30

51.3369 K S 1
KI

SEATWORK Problem 2.18


Solution:
Solving the two equations
simultaneously. We arrive at:
KS = 10.9747 mol/mL
KI = 8.2387 mol/mL

SEATWORK Problem 2.18


Solution:
Any method can be used to solve for
KM and rmax
*By Lineweaver-Burk Plot
KM 1
1
1

r rmax r max C S

@CI = 0
rmax = 0.1516 mol/mLmin R = 0.9996
KM = 11.7725 mol/mL

SEATWORK Problem 2.18


Solution:
@ CI = 7.58 mol/mL
rmax = 0.1463 mol/mLmin
0.9996
KM C
=I =
19.5076
@
30.30 mol/mL
rmax = 0.1406 mol/mLmin
0.9997
KM = 45.1406 mol/mL

R=

R=

SEATWORK Problem 2.18


Solution:
It is observed from the values of KM
and rmax that the inhibition is

CI

given by:
K MI K S 1K
MI is
COMPETITIVE. Hence,

KI

Making use of the values solved for K MI:


KMI = 19.5076 mol/mL @ CI = 7.58 mol/mL
KMI = 45.1406 mol/mL @ CI = 30.30 mol/mL

SEATWORK Problem 2.18


Solution:
Making use of the values solved for KMI:
KMI = 19.5076 mol/mL @ CI = 7.58
mol/mL
KMI =
45.1406
mol/mL
@ CI and
= 30.30
We
now
have two
equations
two

unknowns
by:
7.58
mol/mL 19given

.5076 K 1
S

K I

30.30

45.1406 K S 1
KI

SEATWORK Problem 2.18


Solution:
Solving the two equations
simultaneously. We arrive at:
KS = 10.9557 mol/mL
KI = 9.7107 mol/mL

SEATWORK Problem 2.18


Solution:
Any method can be used to solve for
KM and rmax
*By Eadie-Hofstee Plot
r rmax K M

r
CS

@CI = 0
rmax = 0.1545 mol/mLmin R = 0.9976
KM = 12.1869 mol/mL

SEATWORK Problem 2.18


Solution:
@ CI = 7.58 mol/mL
rmax = 0.1512 mol/mLmin
0.9970
KM C
=I =
20.4919
@
30.30 mol/mL
rmax = 0.1523 mol/mLmin
0.9931
KM = 49.7729 mol/mL

R=

R=

SEATWORK Problem 2.18


Solution:
It is observed from the values of KM
and rmax that the inhibition is
KMI
C I is given
COMPETITIVE. Hence,

K MI K S 1
KI

by:
Making use of the values solved for KMI:
KMI = 20.4919 mol/mL @ CI = 7.58 mol/mL
KMI = 49.7729 mol/mL @ CI = 30.30 mol/mL

SEATWORK Problem 2.18


Solution:
Making use of the values solved for
KMI:
KMI = 20.4919 mol/mL @ CI = 7.58
mol/mL
We
now have two equations and two
KMIunknowns
= 49.7729
mol/mL
@ CI = 30.30

given
by:
7.58

20.4919 K S 1
KI
mol/mL

30.30

49.7729 K S 1
KI

SEATWORK Problem 2.18


Solution:
Solving the two equations
simultaneously. We arrive at:
KS = 10.7230 mol/mL
KI = 8.3203 mol/mL

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
rmaxC S

dt
K M CS
Rearranging, and integrating
yields
CS
t
K M CS
dCS rmaxdt

CS

CS 0

Integrated Form:

CS0

C S0 C S rmaxt
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
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

Schematic Diagram of Plug-Flow Enzyme


Reactor

Linear Form of Prior Given Eqn:


C S 0 C S

ln

C S0

C
S

K M rmax

t
C S0

ln

C
S

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 + Generation =
Accumulation
dCS
FC S0 FC S rSV 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
rmaxC S
F as: 1
D
V
C S0 C S K M C S

where:
D = dilution ratio
Rearranging the equation above gives the
linear relationship
CS K M

C S
rmax
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 C S 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 S

C S rmaxt

CS, mol/m3

t, min

250

0.8646

200

1.8109

150

2.8863

100

4.1972

50

6.0835

---------

Problem 2.6

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

ln

K M rmax

t
C S0

C
S

CS, mol/m3

ln

C
S

t, min

By LR:
KM = -200.0639 mol/m3
rmax = 100.0165
t/ln(CS0/CS)
(CS0
mol/m3min

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:

CS K M

rmaxC 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

Problem 2.7
a. What is the maximum reaction rate?
b. What is the concentration of
substrate after 15 minutes
CS = 2.476210 4 mol/L

Problem 2.8
A substrate is converted to a product by the
catalytic action of an enzyme. Assume the
Michaelis-Menten 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

F = 10 L/h
CS0 = 10
mol/L

KM = 0.03 mol/L
rmax = 13
mol/Lmin

CS = ?
95% conversion

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

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 tworeactor system more efficient than one reactor

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 ,CpH
is equal to pK. For
A
A
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

k 2 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
dCE

kd CE
dt

C E C E0 e kd t

Denaturation constant, kd, varies with


temperature according to the
Ea / RT
Arrhenius eqn k d Ad e

Effect of Temperature
Consequently
r Ae Ea / RT C E0 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

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, air-liquid interface, and some other
effects which are not fully understood.

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

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:
k1 = 7.78107 at T1 = 273 K
k2 = 3.46105 at T2 = 298 K

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