CHAPTER 2: ENZYME KINETICS

2.3 In some enzyme- catalyzed reaction, multiple complexes are involved as follows:
S + E ( ES )1
( ES )1 ( ES )2
( E )2 P + E
REQUIRED: Develop a rate expression using
a. Michaelis Menten approach
b. The Briggs Haldane approach
SOLUTION :
rp=

dcs dcp
=
=ksCesk 4 CpCe
dt
dt

Ceo = Ce = Ces ; Ce = Ceo Ces

rp = k3Ces k4CpCeo + k4CpCes
rp = ( k3 + k4 ) Ces k4CpCeo
k1 CsCe = k2Ces
k1(Cs)(Ceo-Ces) = k2Ces
k1CsCeo-k1CsCes=k2Ces
k2Ces + k1CsCes = k1CsCeo

Ces=

Ces=

k 1CsCeo
k 2+k 1 Cs
CeoCs
k2
+Cs
k1

rp=( k 3+k 4 Cp )

CeoCs
k2
+Cs
k1

- k4Ceo

( kk 21 Cs )(k 4 CpCeo)

( ks+ k 4 Cp ) (CeoCs )
rp =

k2
+Cs
k1

k 3CeoCs+k 4 CeoCpCs
rp=

k 2k 4 CpCeo
k 4 CpCesCs
k1

k2
+Cs
k1
k 2 k 4 CpCeo
k1
k2
+Cs
k1

k 3CeoCs
rp=

Ceo k 3 cs
rp=

k 2k 4
Cp
k1

k2
+Cs
k1

Ce=Ceo=Ces
K1Cs ( Ceo-Ces) = k2Ces
K2Ces + k1CsCes = k1CsCeo
k 1 CsCeo
k2
+Cs
k1
CsCeo
k2
+ Cs
k1
ANSWER
k 3 CeoCs rmaxCx
=
k2
Km+Cs
+Cs
k1

The Briggs Haldane

dcs dcp
=
=k 3 Ces
dt
dt
Rate determining equation:
dcs
=k 1 CsCek 2 Cesk 3Ces=0
dt
Ceo = Ce + Ces
K1Cs ( Ceo-Ces )=k2Ces-k3Ces
K1CsCeo-k1CsCes-k3 ; Ces=0
K1CsCes+k2Ces+k3Ces = k1CsCeo
Ces=

Ces=

k 1 CsCeo
k 1 Cs+k 2+k 3
CsCeo
k 3+k 2
+ Cs
k1

dcs dcp
k 3 CsCeo
=
=
dt
dt
k 3+ k 2
+Cs
k1

rp=

rmaxCs
KmCs

CHAPTER 2: ENZYME KINETICS

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 Concentration
mol/L
0.0032
0.0049
0.0062
0.0080
0.0095

Initial Reaction Rate

mol/Lmin
0.111
0.148
0.143
0.166
0.200

Evaluate the Michaelis-Menten kinetic parameters by employing (a) the Langmuir plot, (b)
the Lineweaver-Burk plot, and (c) the Eadie-Hofstee plot.
Given: *refer to table
Required: Michaelis-Menten kinetic parameters
Solution:
a. Langmuir
CS K M 1
=
+
CS
r
r max r max
x, CS
0.0032
0.0049
0.0062
0.0080
0.0095

y, CS/r
0.0032/0.111
0.0049/0.148
0.0062/0.143
0.0080/0.166
0.0095/0.200

b. Lineweaver-Burk
1 1
KM 1
=
+
r r max r max CS

rmax = 0.3018 mol/Lmin

KM = 5.7721 x 10-3 mol/L

x, 1/CS
1/0.0032
1/0.0049
1/0.0062
1/0.0080
1/0.0095

y, 1/r
1/0.111
1/0.148
1/0.143
1/0.166
1/0.200

rmax = 0.2752 mol/Lmin

KM = 4.7303 x 10-3 mol/L

y, r
0.111
0.148
0.143
0.166
0.200

rmax = 0.2645 mol/Lmin

KM = 4.2731 x 10-3 mol/L

c. Eadie-Hofstee
r = r max - K M
x, r/CS
0.111/0.0032
0.148/0.0049
0.143/0.0062
0.166/0.0080
0.200/0.0095

r
CS

CHAPTER 2: ENZYME KINETICS

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 the reaction and found that 10
percent of the initial substrate was consumed in 5 minutes. The initial substrate concentration is
3.4x10-4 mol/L. Assume that the reaction can be expressed by the Michaelis-Menten kinetics.
a. What is the maximum reaction rate?
b. What is the concentration of the substrate after 15 minutes?
Given:
KM = 0.01 mol/L

@t=5 minutes

Cso= 3.4x10-4 mol/L

10 percent was consumed

Required: a.) rmax

b.) Cs @t=15 minutes

Solution:
K M ln

0.01

Cso
+(CsoCs)=r max t
Cs

mol
1
mol
ln
+ (10.9 ) ( 3.4 x 104 )
=r max ( 5 x 60 ) s
L
0.9
L

r max = 3.6254x10-6 kmol/m3 s

@t= 15 minutes
K M ln

Cso
+(CsoCs)=r max t
Cs

0.01

mol 3.4 x 104

mol
kmol
ln
+ ( 3.4 x 104Cs )
=(3.6254 x 106
) ( 15 x 60 ) s
L
Cs
L
m3 s

Cs= 2.4762 x10-4 mol/ L

CHAPTER 2: ENZYME KINETICS

2.8 A substrate is converted to a product by the catalytic action of an enzyme. Assume that the
Michaelis-Menten kinetic parameters for enzyme reaction are:
KM = 0.03 mol/L
rmax= 13 mol/ L min
a. What should be the size of a steady-state CSTR to convert 95 percent of incoming
substrate (Cso= 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 part (a)?

Given:
KM = 0.03 mol/L
rmax= 13 mol/ L min
Cso= 10 mol/L
F= 10 L/h
Sol'n:
a)
F 1
rmax Cs
= =
V (CsoCs)(Km +Cs)

Req'd:
a)VCSTR
b) VPFR

V=

F
rmax Cs
(CsoCs)(Km+Cs)

( 10hL )( 601 hrmin )

mol
(13 Lmin
)(0.05 10 molL )

V=

10 mol 0.5 mol 0.03 mol 0.5 mol

)(
+
)
L
L
L
L

VCSTR=0.1291 L

b)
CsoCs
t
=Km+rmax (
)
Cso
Cs
ln (
)
ln
Cs
Cso

( )

100.5
t
=( 0.03 ) +(13)(
)
10
10
ln
ln
0.5
0.5

( )

( )

t = 0.7377 min
t=V/F
V=Ft
V=(0.7377 min)(1 h/ 60 min)(10 L/h)
VPFR = 0.1229 L

CHAPTER 2: ENZYME KINETICS

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

grams
liter

g
Rmax = 7 Lmin
If we operate two 1-L CSTR n series at steady state, wht will be the concentration of substrate
leaving the second reactor? The flow rate is 0.5 L/min. The inlet substrate concentration is 50g/L
and the enzyme concentration in the two reactors is maintained in the sa 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?
GIVEN:
Cso
50g/L
Km= 10g/L

rmax= 7g/L-min
F= 0.5 L/min
REQUIRED:
a. Cs2
b. Is two reactor more efficient than 1 reactor with volume = 2L
Cs=-km +

rmax Cs
CsoCs

For the first reactor solve Cs1

( 7Lg ) (Cs 2) ( 0.51lg )

min

Cs1= (-10g/L) +

50 g
Cs1
L

Cs1= 38.8650g/L
At the second reactor ; cso =38.8650g/L

( 7Lg ) (Cs 2) ( 0.51lg )

Cs2= (-10g/L) +

min
38.8650Cs 2

Cs2= 28.50120g/L
Which is more efficient
% conversion =

CsoCs
x 100
Cso

%conversion =

5028.5012
x 100
50

%conversion = 42.9976 % (for 2 CSTR IN SERIES)

For 1 CSTR with volume =2L

( 7Lg ) (Cs 2) ( 0.52lg )

Cs= (-10g/L) +

min
50 g
Cs1
L

Cs= 29.1517 g/L

%conversion=

5029.1517
x 100
50

Conversion = 41.6969 %% ( for 1 CSTR with 2L volume)

CHAPTER 2: ENZYME KINETICS

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), 1.5 x 10 -7
mol/L and obtained the following data:
Substrate Concentration
(mol/L)
0.0032
0.0049
0.0062
0.0080
0.0095

Initial Reaction Rate

Absence of Prostigmine
0.111
0.148
0.143
0.166
0.200

a. Is prostigmine competitive or noncompetitive inhibitor?

Rate (mol/L.min)
Presence of Prostigmine
0.059
0.071
0.091
0.111
0.125

b. Evaluate the Michaelis-Menten kinetic parameters in the presence of inhibitor by

employing the Langmuir plot.
0.08
f(x) = 2.99x + 0.05

0.07
0.06
0.05

Cs/r

f(x) = 3.31x + 0.02

0.04
0.03
0.02
0.01
0

0.01

0.01

0.01

0.01

Cs
Linear ()

a. Prostigmine is a competitive inhibitor.

b. Rmax= 1/m = 0.3346 (with inhibitor concentration)
Rmax= 1/m = 0.3018 (no inhibitor concentration)
Km = bRmax = 5.7644x10-3
KI = bRmax = 0.0164

Linear ()

0.01

0.01

CHAPTER 2: ENZYME KINETICS

2.17 The initital 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):
Given:
Substrate Concentration
Initial Reaction Rate
mol/L
mol/L min
6.7
0.30
3.5
0.25
1.7
0.16
a. Calculate the Michaelis-Menten constants of the above reaction.
b. When the inhibitor was added, the initial reaction rate was decreased as follows:
Substrate
mol/L
6.7
3.5
1.7

Inhibitor
mol/L
146
146
146

Initial Reaction Rate

mol/L min
0.11
0.08
0.06

Is this competitive inhibition or noncompetitive inhibition? Justify your answer by showing the
effect of the inhibitor graphically. [Contributed by Professor Gary F. Bennett, The university of
Toledo, Toledo, OH]
Required: MM constants

Without Inhibitor

With inhibitor

a) Langmuir
C s km
1
=
+
(C )
r r max r max s

a) Langmuir
C s km
1
=
+
(C )
r r max r max s

r = 0.9968 mol/L-min
r(max) = 0.4215 mol/L-min
km = 3.6317 mol/L
b) Lineweaver Burk
k
1
1
1
=
+ m ( )
r r max r max C s

r = 0.9916 mol/L-min
r(max) = 0.1567 mol/L-min
km = 2.9807 mol/L
b) Lineweaver Burk
1
1 km 1 1
=
+
( )
r r max r max C s

r = 0.9961 mol/L-min

r = 0.9876 mol/L-min

r(max) = 0.4511 mol/L-min

km = 3.0566 mol/L
c) Eadie Hofstee
r
r=r max +k m ( )
Cs

r(max) = 0.1416 mol/L-min

km = 2.3613 mol/L
c) Eadie Hofstee
r
r=r max +k m ( )
Cs

r = 0.9781 mol/L-min
r(max) = 0.4336 mol/L-min
km = 2.8096 mol/L

r = 0.9564 mol/L-min
r(max) = 0.1457 mol/L-min
km = 2.5083 mol/

Therefore, Langmuir isotherm best fit the data with r = 0.9968 for withoutinhibitor.

CHAPTER 2: ENZYME KINETICS

2.18 The enzyme, cathepsin, hydrolyzes L-glutamyl-L-tyrosine to carbobenzoxy-L-glutamic 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
mole/mL

Inhibitor
mole/mL

Rate of CO2 Generation

mole/mL.min

4.7
4.7
4.7
10.8
10.8
10.8
30.3
30.3
30.3

0
7.57
30.30
0
7.58
30.30
0
7.58
30.3

0.0434
0.0285
0.0133
0.0713
0.0512
0.0266
0.1111
0.0909
0.0581

Calculate (a) the value of Michaelis-Menten constants of the enzyme, Ks, and (b) the dissociation
constant of enzyme-inhibitor complex, KI.
600
500

f(x) = 6.41x + 329.1

R = 0.99

400
300

f(x) = 6.51x + 137.08

f(x)
R ==16.37x + 80.2
R = 1

200
100
0

10

a. @ I=0
Rmax = 1/m = 0.1569
Ks = bRmax = 12.5839

15

20

25

30

35

@ I = 30.30
Rmax = 1/m = 0.1560
KI = bRmax = 51.3368

@ I= 7.58
Rmax = 1/m = 0.1537
KI = bRmax = 21.0720
CHAPTER 2: ENZYME KINETICS ; ADDITIONAL PROBLEM
https://ww2.chemistry.gatech.edu/~lw26/bCourse_Information/3511/stud_comp/chap12_17.pdf
The following data were obtained for the reaction A B, catalyzed by the enzyme Aase. The
reaction volume was 1mL and the stock concentration of A was 5.0mM. Seven separate reactions
were examined, each containing a different amount of A. The reactions were initiated by adding
2.0L of a 10M solution of Aase. After 5 minutes, the amount of B was measured.
Reaction
1
2
3
4
5
6
7

Volume of A
added(L)
8
10
15
20
40
60
100

Amount of B present
at 5 minutes (nmoles)
26
29
39
43
56
62
71

(a) Calculate the initial velocity of each reaction (in units of M.min-1)
(b) Determine the KM and Vmax of Aase from a Lineweaver-Burk plot.
(c) Calculate kcat.
SOLN:
(a) o = (26nmol/5min) / (1.0mL) x (103 mL/L) x (.001 mol/1nmol) = 5.2 M.min-1
Reaction
1
2
3
4
5
6
7

o(M.min-1)
5.2
5.8
7.8
8.6
11.2
12.4
14.2

(b) Calculate [S] for each reaction

[A] = (.008mL)(5mM)(1mL) x (1000 M/1 mM) = 40 M
Reaction

[S] M

(x) 1/[S] M-1

(M.min-1)

1
2
3
4
5
6
7

40
50
75
100
200
300
500

0.025
0.02
0.0133
0.010
0.005
0.0033
0.002

5.2
5.8
7.8
8.6
11.2
12.4
14.2

y-int = 1/vmax = 0.06

Vmax = 16 M/min
x-int = -1/KM = 1/0.012
KM = 83 M
(c) Calculate [E]T = (0.002mL)(10 M Aase) / 1mL = 0.02 M
Kcat = Vmax / [E]T = (16 M/min) / 0.02 M
Kcat= 800 /min

(y) 1/ (min-1/
M)
0.192
0.172
0.128
0.0116
0.089
0.081
0.070

CHAPTER 2: ENZYME KINETICS ; ADDITIONAL PROBLEM

The statin drug lovastian helps lower cholesterol level by acts as competitive inhibitor on the
HMG-CoA reductase enzyme, which normally catalyzes an early step in the biosynthesis
pathway cholesterol
Required:
a) On a single graph, sketch the Michaelis-menten plot for HMG-CoA reductase in the presence
and absence of lovastatin, clearly labeling Km, and Vmax.
b) On a single graph, sketch the lineweaver-Burke (double reciprocal) plot for HMGCoA
reductase in the presence and absence of lovastatin. Clearly indicating how you could determin
Km, kcat and Vmax.
Solution:
a)

b)

CHAPTER 2: ENZYME
KINETICS ; ADDITIONAL PROBLEM
Pesticide inhibition on enzyme has been reported, which caused the enzyme activity to reduce.
The collected data with and without inhibition are presented below. Determine the type of
inhibition and the KI for the inhibitor.
[S], M
Rate [I=0],
M/min103
Rate
[I=20nM],
M/min103

3.3010-4
56

5.0010-4
71

6.7010-4
88

1.6510-3
124

2.2110-3
149

37

47

61

103

125

Assuming Lineweaver-Burk Equation:

1 Km 1
1
=
+
V V max S V max

[I=0]: Line 1: y=4.320010-9x+5.003310-6

1
V max

=5.003310-6

V max = 199,868.0871 M/min

Km
-9
V max =4.320010
K m = 8.634310-4 M

[I=20nM]: Line 2: y=7.460510-9x+5.169010-6

1
-6
V max =5.169010

V max = 193,461.0176 M/min

K mapp
-9
V max =7.460510

K mapp = 1.443310-3 M

[ ]

K m 1+

I
=K mapp
; 8.634310-4 M
KI

K I = 2.978010-8 M

Type of Inhibition: COMPETITIVE

K I = 2.978010-8 M

20 109 M
1+
= 1.443310-3 M
KI

CHAPTER 2: ENZYME KINETICS ; ADDITIONAL PROBLEM

A certain reaction has an activation energy of 125 kJ/mol. The rate is 0.33/s at 55C. Determine
the value of the specific rate constant at 100 C.
GIVEN: Ea=125 kJ/mol
@T1=55 C ; K55 C = 0.33

REQUIRED: @T2=100 C ; K100 C = ?

SOLUTION:
K =A e

E a
RT

@T1=55 C:
0.33= A e

125 kJ /mol
8.314 kj
(55+273)K
kmol .K

A=2.60991019
@T2=100 C:
K100C = (2.60991019) e

K100C =82.8182/s

125kJ / mol
8.314 kj
(100+273)K
kmol .K

CHAPTER 2: ENZYME KINETICS ; ADDITIONAL PROBLEM

The enzyme carboxypeptidase catalyzes the hydrolysis of peptides. The following results were
obtained when the rate of enzymolysis of CBGP was monitored without inhibition at [CBGP]0=
0.713 mol/dm3.
CBGP mol
,
102 dm 3
Rate,
mol
dm3 . s

1.25

3.84

5.81

7.13

0.398

0.649

0.859

1.00

When 2.010-3 mol/dm3 phenyl butyrate ion was added to the solution, the results were:
CBGP mol
,
102 dm 3
Rate,
mol
dm3 . s

1.25

2.50

4.00

5.50

0.172

0.301

0.344

0.548

In a separate experiment, the effect of 5.010-2 mol/dm3 benzoate ion was monitored and the
results were:
CBGP mol
,
102 dm 3
Rate,
mol
dm3 . s

1.75

2.50

5.00

10.00

0.183

0.201

0.231

0.246

Determine the type of inhibition and KI for phenyl butyrate and benzoate ion.

SOLUTION:
Assuming Lineweaver-Burk Equation:
Line 1: without inhibition

1 Km 1
1
=
+
V V max S V max

y=0.8126+0.0216x
V max = 1.2306 mol/dm3.s
K m = 0.0266 mol/dm3

Line 2: with phenyl butyrate ion

y=1.0158+0.0601x
V max = 0.9845 mol/dm3.s
K m = 0.0592 mol/dm3
Line 3: with benzoate ion
y=3.7517+0.0300x
V max = 0.2665 mol/dm3.s
K m = 8.0228 mol/dm3

TYPE OF INHIBITION:

Phenyl Butyrate Ion: COMPETITIVE

Benzoate Ion: UNCOMPETITIVE

FOR PHENYL BUTYRATE ION:

[ ]

K m 1+

KI

I
2.0 103
=K mapp
0.0266
1+
=0.0592
;
KI
KI

= 1.636910-3 mol/dm3

FOR BENZOATE ION:

V maxapp=

KI

V max
I
1+
KI

; 0.2665

= 0.0138 mol/dm3

1.2306
5.0 102
1+
KI