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 (min) Glucose Concentration (µmol/mL)

1 0.05
5 0.23
10 0.38
15 0.52
30 1.03

a. What is the activity of the β-glucosidase in units/mL of enzyme solution and in units/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?

Concentration vs Time
1.5

1
[S]

0.5 y = 0.033x + 0.0391

R² = 0.9973
0
0 5 10 15 20 25 30 35
t

a. Vt = Vcellobiose + Vbuffer solution + Venzyme 1.65 mumol/min

Vt * m  =
= 5 mL + 44 mL + 1 mL 1 mL of enzyme
= 50 mL 1.65 units/mL of enzyme = 165
Plotting [S] vs time yields an units/mg protein
equation with a slope of m = 0.033
(mumol/mL-min). therefore, the b. Initial reaction rate is the slope of the
activity is equation. Thus, reaction rate is
Vt * m = (50 mL)(.033) m=0.033 mumol/mL-min
= 1.65 mumol/min
2.5 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 min)

(mol/L)
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, (c) the Eadie-Hofstee plot, and (d) non-linear regression procedure.

(a) Langmuir Plot

Langmuir
LANGMUIR PLOT
0.06 Cs/r
Cs
0.05
0.028829
0.04 0.0032
CS/R

0.03 y = 3.3133x + 0.0191 0.033108

R² = 0.8837 0.0049
0.02
0.043357
0.0062
0.01
0.048193
0 0.008
0 0.002 0.004 0.006 0.008 0.01
CS 0.0475
0.0095
(b) Lineweaver-Burk Plot

Lineweaver-Burk
LINEWEAVER-BURK PLOT
10 1/Cs 1/r
9
8
7
312.5 9.009009
6
5 204.0816 6.756757
1/R

y = 0.0172x + 3.6342
4 R² = 0.9146
3 161.2903 6.993007
2
1
125 6.024096
0
0 50 100 150 200 250 300 350
1/CS 105.2632 5

(c) Eadie-Hofstee Plot

Eadie-Hofstee

EADIE-HOFSTEE PLOT r/Cs r

0.25
34.6875 0.111
0.2
30.20408 0.148
0.15
R

23.06452 0.143
0.1
y = -0.0043x + 0.2645
R² = 0.6584
0.05 20.75 0.166

0 21.05263 0.2
0 5 10 15 20 25 30 35 40
R/CS
(d) Nonlinear Regression

Nonlinear Regression
0.25

0.2

0.15

0.1

0.05

0
0 0.002 0.004 0.006 0.008 0.01

Vmax = 0.2