Evaluation of Michaelis-Menten Parameters

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.

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

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
Slope: KM/rmax
Intercept: 1/rmax
EADIE-HOFSTEE DIAGRAM
Also known as:
Woolf-Eadie-Augustinsson-Hofstee
Eadie-Augustinsson
r rmax K M
r
CS

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

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

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
w/ inhibitor
f(x) = - 0.01x + 0.26
0.1
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
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
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
w/ inhibitor
30
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
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
w/ inhibitor
0.15
0.1
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.

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

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]

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

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

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

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

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

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

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

Solution:
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

Solution:
@ CI = 7.58 mol/mL
0.9996
KM C
=I =
19.5076
@
30.30 mol/mL
0.9997
KM = 45.1406 mol/mL
R=
R=

Solution:
CI
given by:
K MI K S 1K
MI is
COMPETITIVE. Hence,
KI
Making use of the values solved for K MI:


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

Solution:
KS = 10.9557 mol/mL
KI = 9.7107 mol/mL

Solution:
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

Solution:
@ CI = 7.58 mol/mL
0.9970
KM C
=I =
20.4919
@
30.30 mol/mL
0.9931
KM = 49.7729 mol/mL
R=
R=

Solution:
KMI
C I is given
COMPETITIVE. Hence,
K MI K S 1
KI
by:
Making use of the values solved for KMI:

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

Solution:
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.

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

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.
Figure below depicts the variation of
reaction rate with temperature and
the presence of an optimal
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
where
Ea = activation energy
CE = active enzyme concentration
Plot of ln(r) versus 1/T results in a line
with slope Ea/R
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
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

Evaluation of Michaelis-Menten Parameters

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Evaluation of Michaelis-Menten Parameters

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Kinetic parameters for several

Kinetic parameters for several

Carbobenzoxy-Ltyrosine ethyl ether

Kinetic parameters for several

The most straightforward way is to

The asymptote for r will be rmax and

The Michaelis-Menten equation is

This equation was given by Langmuir

Slope: 1/rmax and intercept: KM/rmax

Refer to Figure 2.6, p. 25, James Lee

3. Estimate the kinetic parameters by

a. Evaluate the Michaelis-Menten kinetic

a. Evaluate the Michaelis-Menten kinetic

w/o prostigmine: w/ prostigmine

f(x) = 3.31x + 0.02

w/o prostigmine: w/ prostigmine

f(x) = 0.02x + 3.63

w/o prostigmine: w/ prostigmine

Linear (w/ inhibitor)

f(x) = 6.38x + 19.02

Linear (w/ inhibitor)

f(x) = 2.37x + 6.24

f(x) = 16.68x + 7.06

Linear (w/ inhibitor)

f(x) = - 2.81x + 0.43

Linear (w/ inhibitor)

f(x) = - 2.51x + 0.15

b. Since KM did not change greatly relative to

SEATWORK Problem 2.18

SEATWORK Problem 2.18

SEATWORK Problem 2.18

SEATWORK Problem 2.18

SEATWORK Problem 2.18

SEATWORK Problem 2.18

SEATWORK Problem 2.18

SEATWORK Problem 2.18

Making use of the values solved for K MI:

SEATWORK Problem 2.18

SEATWORK Problem 2.18

SEATWORK Problem 2.18

SEATWORK Problem 2.18

SEATWORK Problem 2.18

Making use of the values solved for K MI:

SEATWORK Problem 2.18

SEATWORK Problem 2.18

SEATWORK Problem 2.18

SEATWORK Problem 2.18

SEATWORK Problem 2.18

SEATWORK Problem 2.18

SEATWORK Problem 2.18

Enzyme Reactor and Simple Kinetics

Batch / Plug-Flow Reactor (SteadyState)

Schematic Diagram of a Batch Reactor

Plug-flow enzyme reactor (tubular-flow enzyme

Schematic Diagram of Plug-Flow Enzyme

Linear Form of Prior Given Eqn:

Continuous Stirred-Tank Reactor

Continuous Stirred-Tank Reactor

Continuous Stirred-Tank Reactor

b. Assume that you obtained the C S versus t

a. What is the activity of the -glucosidase in

Other Influences on Enzyme

The typical relationship b/n the rxn velocity and