GROUP 6

Daquiog, Justine
Decena, Clarissa
Mamba, Rhea
Singson, Mariella

PROBLEM 2.1
In order to measure the enzyme activity and the initial rate of reaction, 5 mL of cellobiose
(100mumol/mL) and 44 mL of buffer solution were placed in a stirred vessel. The reaction was
initiated by adding 1 mL of enzyme (beta-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 the reaction mixture
and its glucose content was measured. The results were as follows:
TIME GLUCOSE CONCENTRATION
(min) (µmol/mL)
1 0.05
5 0.23
10 0.38
15 0.52
30 1.03

REQUIRED:
a. What is the activity of the β-glucosidase in units/mL of enzyme solution and in umts/mg
protein? A unit is defined as the enzyme activity which can produce Imumol of product per
minute.
b. What is the initial rate of reaction?
SOLUTION:
a. Total Volume = 44 + 5 + 1 = 50 mL

Concentration vs Time
1.2

y = 0.033x + 0.0391
1
R² = 0.9973
0.8
C

0.6

0.4

0.2

0
0 5 10 15 20 25 30 35
t

Based from the graph above;

y = 0.033x + 0.0391
m = 0.033mumol/ml.min
0.033*50=1.65 mumol/min
Activity of the β-glucosidase,
1.65mumol / min
A1 =
.1mg / ml * .1ml
A1 =165 units/mg protein

1.65mumol / min
A2 =
1mlenzyme

A2 = 1.65 units/ml of enzyme

b. m = Initial rate of reaction

m = 0.033mumol/mL.min
 PROBLEM 2.15
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 Initial Reaction Rate (mol/L-min) Cs/r Cs/r

Concentration, Rate Absence of Presence of (Uninhibited) (Inhibited)
Cs (mol/L) Prostigmine Prostigmine
0.0032 0.111 0.059 0.028828829 0.05423729
0.0049 0.148 0.071 0.033108108 0.06901408
0.0062 0.143 0.091 0.043356643 0.06813187
0.008 0.166 0.111 0.048192771 0.07207207
0.0095 0.2 0.125 0.0475 0.076

REQUIRED:

a. Is prostigmine competitive or noncompetitive inhibitor?

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

the Langmuir Plot.

SOLUTION:

a. Prostigmine as competitive or noncompetitive inhibitor

Langmuir Plot of Hydrolysis of Acetylcholine

0.09
0.08 y = 2.9883x + 0.0489
0.07
0.06
0.05 y = 3.3133x + 0.0191
Cs/r 0.04 Without Inhibitor
0.03 With Inhibitor
0.02
-KMI -KM 0.01
0
-0.02 -0.015 -0.01 -0.005 -0.01 0 0.005 0.01 0.015
Cs

Based from the graph, the values of Km with and without the presence of inhibitor is not equal.
Therefore, prostigmine is a competitive inhibitor.
b. Langmuir Equation (With inhibitor):

Cs 1 K
 Cs  m
r rmax rmax

y  2.9883x  0.0489

1
 2.9883
rmax

mol
rmax  0.3346
L  min
K mi
 0.0489
rmax

mol
K mi  0.01636
L
PROBLEM 2.19
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. BioI.Chern.,
233, 159, 1958):

Substrate Concentration Initial Reaction Rate (µmol/L- 1/Cs 1/r

(µmol/L) min)

6.7 0.3 0.149254 3.333333

3.5 0.25 0.285714 4
1.7 0.16 0.588235 6.25

REQUIRED:
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 Concentration Inhibitor Initial Reaction 1/r

(µmol/L) (µmol/L) Rate (µmol/L-
min)
6.7 146 0.11 9.090909
3.5 146 0.08 12.5
1.7 146 0.06 16.66667
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]

SOLUTION:
a. Michaelis-Menten constants of the below reaction

Substrate Concentration Initial Reaction Rate (µmol/L- 1/Cs 1/r

(µmol/L) min)

6.7 0.3 0.149254 3.333333

3.5 0.25 0.285714 4
1.7 0.16 0.588235 6.25

Lineweaver-Burk Plot of Enzymatic Cleave

of Deoxyguanosine Triphosphate
7

6
y = 6.7758x + 2.2168
5
1/r

1
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
1/Cs

Lineweaver-Burk Equation:
1 Km 1 1
 
r rmax Cs rmax

y  6.7758 x  2.2168

1
 2.2168
rmax
 mol
rmax  0.451
L  min
Km
 6.7758
rmax

mol
K m  3.0566
L

b. Plotting the graph of the reaction with and without inhibitor using Lineweaver-Burk Equation

Lineweaver-Burk Plot of Enzymatic Cleavage of

Deoxyguanosine Triphosphate
18
y = 16.68x + 7.0637
16
R² = 0.9754
14
12
10
1/r 8
6
4 y = 6.7758x + 2.2168
2 R² = 0.9922
0
-0.6 -0.4 -0.2 -2 0 0.2 0.4 0.6 0.8

1/Cs
Without inhibitor WIth inhibitor

LINEWEAVER-BURK EQUATION
With Inhibitor Without Inhibitor
rmax Km rmax Km
0.141569 2.361369 0.4511007 3.056568

Based from the graph, the values of Km (with and without inhibitor) are almost the same. Moreover,
the y-intercepts, rmax of the Lineweaver-Burk for both inhibited and uninhibited are not equal.
Therefore, it is a noncompetitive inhibition.
PROBLEM 2.20
The effect of an inhibitor on an enzyme reaction was studied by measuring the initial rates at three
different initial inhibitor concentrations. The obtained Michaelis-Menten kinetic parameters are as
follows:

Inhibitor rmax KM
µmol/L µmol/L min µmol/L
0 0.70 5
2 0.20 5
4 0.11 5
6 0.08 5

REQUIRED:
a. Write the kinetic model for this enzyme reaction.
b. Derive the rate equation. State your assumptions for any simplification of the equation.
c. Estimate the value of inhibition kinetic parameter.

SOLUTION:
a. Kinetic model for this enzyme reaction
Since the given has constant KM shown in the table given table, then the enzyme reaction
is non-competitive inhibition reaction.
The kinetic model would be:
K1

E +S ⇔
K
ES
2

K3

E+I ⇔
K4
EI
K5

EI + S ⇔
K6
EIS
K7

ES + I ⇔
K
ESI
8

K9

ES → E + P
b. Derive the rate equation. State your assumptions for any simplification of the equation.
Assumptions:
 The dissociation constant for the first equilibrium reaction is the same as that of the third
equilibrium reaction.
 The dissociation constant for the second equilibrium reaction is the same as that of the
fourth equilibrium equation.
The two equilibrium reactions,
k2 k
 K S  6  K IS
k1 k5

k4 k
 K I  8  K SI
k3 k7

If the slower reaction, the product formation step, determines the rate of the reaction according to
Michaelis-Menten assumption, the rate can be expressed as:
rp  k9 ES  (1)

The enzyme balance gives

E0   E   ES   EI   ESI  (2)

Divide (1) by (2),

rp k9 ES 
 (3)
E0  E   ES   EI   ESI 
Applied Law of Mass Action

K 2 E S  E S 
KS    ES   (4)
K1 ES  KS

K 4 E I  E I 
KI    EI   (5)
K3 EI  KI

K 8 ES I  ES I 

KS    ESI   (6)
K7 ESI  KI
Substitute (4), (5), (6) into (3),

k9
E S 
rp K

E 0  E   E S   E I   ES I 
KS KI KI

k9
E S 
rp K

E 0  E   E S   E I   E S I 
KS KI KS KI

Eliminate [E],
S 
rp KS

EO  k9 1
S   I   S I 
KS KI KS KI

Substitute rpmax  E 0  k 9

S 
rp KS

rpmax 1
S   I   S I 
KS KI KS KI

Multiply numerator and denominator by K S p

rp

S 
K S I  S I 
K S  S  
r pmax

KI KI

Rearranging
rp

S 
K S I  S I 
 S  
r pmax
KS 
KI KI
rp

S 
r pm ax 
K S  1 
I    S   1  I  
 K I  
 K I 