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 :

−dcs dcp
rp = = = ksCes − k4CpCe
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

k1 CsCeo
Ces =
k2 + k1Cs

CeoCs
Ces =
k2
+ Cs
k1

CeoCs
rp = ( k3 + k4Cp) k2 - k4Ceo
+Cs
k1

k2
(ks+k4Cp)(CeoCs)− ( Cs ) ( k4CpCeo )
k1
rp = k2
+Cs
k1
k2k4CpCeo
k3CeoCs + k4CeoCpCs − − k4CpCesCs
rp = k1
k2
+ Cs
k1
 k2k4CpCeo
k3CeoCs −
rp = k1
k2
+ Cs
k1

k2k4
Ceo (k3cs − Cp)
rp = k1
k2
+ Cs
k1
Ce=Ceo=Ces

K1Cs ( Ceo-Ces) = k2Ces

K2Ces + k1CsCes = k1CsCeo

k1CsCeo
k2
+ Cs
k1

CsCeo
k2
+ Cs
k1

ANSWER

k3CeoCs rmaxCx
=
k2
+ Cs Km + Cs
k1
The Briggs Haldane

−dcs dcp
= = k3Ces
dt dt

Rate determining equation:

−dcs
= k1CsCe − k2Ces − k3Ces = 0
dt

Ceo = Ce + Ces
K1Cs ( Ceo-Ces )=k2Ces-k3Ces
K1CsCeo-k1CsCes-k3 ; Ces=0
K1CsCes+k2Ces+k3Ces = k1CsCeo

k1CsCeo
Ces =
k1Cs + k2 + k3

CsCeo
Ces =
k3 + k2
+ Cs
k1

−dcs dcp k3CsCeo

= =
dt dt k3 + k2
+ Cs
k1

rmaxCs
rp= 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 Initial Reaction Rate

mol/L mol/L·min
0.0032 0.111
0.0049 0.148
0.0062 0.143
0.0080 0.166
0.0095 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 KM 1
= + CS
r rmax rmax

x, CS y, CS/r
0.0032 0.0032/0.111
0.0049 0.0049/0.148
0.0062 0.0062/0.143
0.0080 0.0080/0.166 rmax = 0.3018 mol/L·min
0.0095 0.0095/0.200 KM = 5.7721 x 10-3 mol/L

b. Lineweaver-Burk
1 1 KM 1
= +
r rmax rmax CS
x, 1/CS y, 1/r
1/0.0032 1/0.111
1/0.0049 1/0.148
1/0.0062 1/0.143
1/0.0080 1/0.166 rmax = 0.2752 mol/L·min
1/0.0095 1/0.200 KM = 4.7303 x 10-3 mol/L
c. Eadie-Hofstee
r
r = rmax - KM
CS
x, r/CS y, r
0.111/0.0032 0.111
0.148/0.0049 0.148
0.143/0.0062 0.143
0.166/0.0080 0.166 rmax = 0.2645 mol/L·min
0.200/0.0095 0.200 KM = 4.2731 x 10-3 mol/L
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 𝐶𝑠𝑜
𝐶𝑠
+ (𝐶𝑠𝑜−𝐶𝑠) = 𝑟𝑚𝑎𝑥 𝑡

mol
0.01 1
ln 0.9 + (1−0.9)(3.4x10−4)
𝑚𝑜𝑙
𝐿
= 𝑟𝑚𝑎𝑥 (5𝑥60)𝑠
L

𝑟𝑚𝑎𝑥 = 3.6254x10-6 kmol/m3 s

@t= 15 minutes

K M ln 𝐶𝑠𝑜
𝐶𝑠
+ (𝐶𝑠𝑜−𝐶𝑠) = 𝑟𝑚𝑎𝑥 𝑡

mol kmol
0.01 ln 3.4𝑥10−4
𝐶𝑠
+ (3.4𝑥10−4−𝐶𝑠)
𝑚𝑜𝑙
𝐿
= (3.6254x10 − 6 m3 s ) (15𝑥60)𝑠
L

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: Req'd:
KM = 0.03 mol/L a)VCSTR
rmax= 13 mol/ L min b) VPFR
Cso= 10 mol/L
F= 10 L/h

Sol'n:
a)
𝐹 1 𝑟𝑚𝑎𝑥 𝐶𝑠
= =
𝑉 𝜏 (𝐶𝑠𝑜 − 𝐶𝑠)(𝐾𝑚 + 𝐶𝑠)

𝐹
𝑉=
𝑟𝑚𝑎𝑥 𝐶𝑠
(𝐶𝑠𝑜 − 𝐶𝑠)(𝐾𝑚 + 𝐶𝑠)

10𝐿 1ℎ𝑟
(
) (60min)
𝑉= ℎ
𝑚𝑜𝑙 𝑚𝑜𝑙
(13 𝐿 𝑚𝑖𝑛) (0.05 × 10 𝐿 )
10𝑚𝑜𝑙 0.5𝑚𝑜𝑙 0.03𝑚𝑜𝑙 0.5𝑚𝑜𝑙
( 𝐿 − 𝐿 )( + 𝐿 )
𝐿

VCSTR=0.1291 L

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

10 − 0.5 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:
grams
Km=10 liter
g
Rmax = 7 L−min

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

rmax Csτ
Cs=-km + Cso−Cs
For the first reactor solve Cs1

7g 1l
( )(Cs2)( 0.5g )
L
min
Cs1= (-10g/L) + 50g
− Cs1
L
Cs1= 38.8650g/L

At the second reactor ; cso =38.8650g/L

7g 1l
( )(Cs2)( 0.5g )
L
min
Cs2= (-10g/L) + 38.8650− Cs2
Cs2= 28.50120g/L

Which is more efficient

Cso−Cs
% conversion = x 100%
Cso

50−28.5012
%conversion = x 100%
50

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

For 1 CSTR with volume =2L

7g 2l
( )(Cs2)( 0.5g )
L
min
Cs= (-10g/L) + 50g
− Cs1
L

Cs= 29.1517 g/L

50 − 29.1517
%conversion = 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 Initial Reaction Rate Rate (mol/L.min)
(mol/L) 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. Is prostigmine competitive or noncompetitive inhibitor?

b. Evaluate the Michaelis-Menten kinetic parameters in the presence of inhibitor by
employing the Langmuir plot.
0.09
0.08
y = 2.9883x + 0.0489
0.07
0.06
0.05 y = 3.3133x + 0.0191
Cs/r

0.04
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
Cs

Series1 Series2 Linear (Series1) Linear (Series2)

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
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
μmol/L μmol/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 Inhibitor Initial Reaction Rate
μmol/L Μmol/L Μmol/L min
6.7 146 0.11
3.5 146 0.08
1.7 146 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 a) Langmuir
𝐶𝑠 𝑘𝑚 1 𝐶𝑠 𝑘𝑚 1
= + (𝐶 ) = + (𝐶 )
𝑟 𝑟𝑚𝑎𝑥 𝑟𝑚𝑎𝑥 𝑠 𝑟 𝑟𝑚𝑎𝑥 𝑟𝑚𝑎𝑥 𝑠

r = 0.9968 μmol/L-min r = 0.9916 μmol/L-min

r(max) = 0.4215 μmol/L-min r(max) = 0.1567 μmol/L-min
km = 3.6317 μmol/L km = 2.9807 μmol/L
b) Lineweaver Burk b) Lineweaver Burk
1 1 𝑘𝑚 1 1 1 𝑘𝑚 1 1
= + ( ) = + ( )
𝑟 𝑟𝑚𝑎𝑥 𝑟𝑚𝑎𝑥 𝐶𝑠 𝑟 𝑟𝑚𝑎𝑥 𝑟𝑚𝑎𝑥 𝐶𝑠

r = 0.9961 μmol/L-min r = 0.9876 μmol/L-min

r(max) = 0.4511 μmol/L-min r(max) = 0.1416 μmol/L-min
km = 3.0566 μmol/L km = 2.3613 μmol/L
c) Eadie Hofstee c) Eadie Hofstee
𝑟 𝑟
𝑟 = 𝑟𝑚𝑎𝑥 + 𝑘𝑚 ( ) 𝑟 = 𝑟𝑚𝑎𝑥 + 𝑘𝑚 ( )
𝐶𝑠 𝐶𝑠

r = 0.9781 μmol/L-min r = 0.9564 μmol/L-min

r(max) = 0.4336 μmol/L-min r(max) = 0.1457 μmol/L-min
km = 2.8096 μmol/L km = 2.5083 μmol/

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 Inhibitor Rate of CO2 Generation

𝜇mole/mL 𝜇mole/mL 𝜇mole/mL.min
4.7 0 0.0434
4.7 7.57 0.0285
4.7 30.30 0.0133
10.8 0 0.0713
10.8 7.58 0.0512
10.8 30.30 0.0266
30.3 0 0.1111
30.3 7.58 0.0909
30.3 30.3 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
y = 6.4106x + 329.1
500
R² = 0.9935
400
y = 6.5053x + 137.08
300 y =R²6.3732x
= 0.9986
+ 80.2
R² = 0.9993
200

100

0
0 5 10 15 20 25 30 35

a. @ I=0 @ I = 30.30
Rmax = 1/m = 0.1569 Rmax = 1/m = 0.1560
Ks = bRmax = 12.5839 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
2.0µL of a 10µM solution of Aase. After 5 minutes, the amount of B was measured.
Reaction Volume of A Amount of B present
added(µL) at 5 minutes (nmoles)
1 8 26
2 10 29
3 15 39
4 20 43
5 40 56
6 60 62
7 100 71

(a) Calculate the initial velocity of each reaction (in units of µM.min-1)
(b) Determine the KM and Vmax of Aase from a Lineweaver-Burk plot.
(c) Calculate kcat.
SOL’N:
(a) νo = (26nmol/5min) / (1.0mL) x (103 mL/L) x (.001 µmol/1nmol) = 5.2 µM.min-1
Reaction νo(µM.min-1)
1 5.2
2 5.8
3 7.8
4 8.6
5 11.2
6 12.4
7 14.2

(b) Calculate [S] for each reaction

 [A] = (.008mL)(5mM)(1mL) x (1000 µM/1 mM) = 40 µM
Reaction [S] µM (x) 1/[S] µM-1 ν(µM.min-1) (y) 1/ν (min-1/
µM)
1 40 0.025 5.2 0.192
2 50 0.02 5.8 0.172
3 75 0.0133 7.8 0.128
4 100 0.010 8.6 0.0116
5 200 0.005 11.2 0.089
6 300 0.0033 12.4 0.081
7 500 0.002 14.2 0.070

y-int = 1/vmax = 0.06

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

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 3.30×10-4 5.00×10-4 6.70×10-4 1.65×10-3 2.21×10-3
Rate [I=0], 56 71 88 124 149
M/min×103
Rate
[I=20nM], 37 47 61 103 125
M/min×103

1 𝐾𝑚 1 1
Assuming Lineweaver-Burk Equation: =𝑉 +𝑉
𝑉 𝑚𝑎𝑥 𝑆 𝑚𝑎𝑥

[I=0]: Line 1: y=4.3200×10-9x+5.0033×10-6

1 𝐾𝑚
=5.0033×10-6 =4.3200×10-9
𝑉𝑚𝑎𝑥 𝑉𝑚𝑎𝑥

𝑉𝑚𝑎𝑥 = 199,868.0871 M/min 𝐾𝑚 = 8.6343×10-4 M

[I=20nM]: Line 2: y=7.4605×10-9x+5.1690×10-6

1
=5.1690×10-6
𝑉𝑚𝑎𝑥

𝑉𝑚𝑎𝑥 = 193,461.0176 M/min

𝐾𝑚 𝑎𝑝𝑝
=7.4605×10-9
𝑉𝑚𝑎𝑥

𝐾𝑚 𝑎𝑝𝑝 = 1.4433×10-3 M
 𝐼 20×10−9 𝑀
𝐾𝑚 [1 + 𝐾 ] = 𝐾𝑚 𝑎𝑝𝑝 ; 8.6343×10-4 M [1 + ] =1.4433×10-3 M
𝐼 𝐾𝐼

𝐾𝐼 = 2.9780×10-8 M

Type of Inhibition: COMPETITIVE

𝐾𝐼 = 2.9780×10-8 M
CHAPTER 2: ENZYME KINETICS ; ADDITIONAL PROBLEM

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

REQUIRED: @T2=100 ºC ; K100 ºC = ?

SOLUTION:
−𝐸𝑎
K =A𝑒 𝑅𝑇
@T1=55 ºC:
−125 𝑘𝐽/𝑚𝑜𝑙
8.314𝑘𝑗
(55+273)𝐾
0.33= A𝑒 𝑘𝑚𝑜𝑙.𝐾

A=2.6099×1019
@T2=100 ºC:
−125 kJ/mol
8.314kj
19 (100+273)K
K100ºC = (2.6099×10 )e kmol.K

K100ºC =82.8182/s
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 1.25 3.84 5.81 7.13
,
10−2 𝑑𝑚3
𝑚𝑜𝑙 0.398 0.649 0.859 1.00
Rate, 𝑑𝑚3 .𝑠

When 2.0×10-3 mol/dm3 phenyl butyrate ion was added to the solution, the results were:
𝐶𝐵𝐺𝑃 𝑚𝑜𝑙 1.25 2.50 4.00 5.50
,
10−2 𝑑𝑚3
𝑚𝑜𝑙 0.172 0.301 0.344 0.548
Rate, 𝑑𝑚3 .𝑠

In a separate experiment, the effect of 5.0×10-2 mol/dm3 benzoate ion was monitored and the
results were:
𝐶𝐵𝐺𝑃 𝑚𝑜𝑙 1.75 2.50 5.00 10.00
,
10−2 𝑑𝑚3
𝑚𝑜𝑙 0.183 0.201 0.231 0.246
Rate, 𝑑𝑚3 .𝑠

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

SOLUTION:
1 𝐾𝑚 1 1
Assuming Lineweaver-Burk Equation: =𝑉 +𝑉
𝑉 𝑚𝑎𝑥 𝑆 𝑚𝑎𝑥

Line 1: without inhibition

y=0.8126+0.0216x
𝑉𝑚𝑎𝑥 = 1.2306 mol/dm3.s
𝐾𝑚 = 0.0266 mol/dm3
Line 2: with phenyl butyrate ion
y=1.0158+0.0601x
𝑉𝑚𝑎𝑥 = 0.9845 mol/dm3.s
𝐾𝑚 = 0.0592 mol/dm3
Line 3: with benzoate ion
y=3.7517+0.0300x
𝑉𝑚𝑎𝑥 = 0.2665 mol/dm3.s
𝐾𝑚 = 8.0228 mol/dm3

TYPE OF INHIBITION:
 Phenyl Butyrate Ion: COMPETITIVE
 Benzoate Ion: UNCOMPETITIVE

FOR PHENYL BUTYRATE ION:

𝐼 2.0×10−3
𝐾𝑚 [1 + 𝐾 ] = 𝐾𝑚 𝑎𝑝𝑝 ; 0.0266 [1 + ] = 0.0592
𝐼 𝐾𝐼

𝐾𝐼 = 1.6369×10-3 mol/dm3

FOR BENZOATE ION:

𝑉𝑚𝑎𝑥 1.2306
𝑉𝑚𝑎𝑥 𝑎𝑝𝑝 = 𝐼 ; 0.2665= 5.0×10−2
1+ 1+
𝐾𝐼 𝐾𝐼

𝐾𝐼 = 0.0138 mol/dm3