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) (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?
1.5 1 ] S [
0.5
y = 0.033x + 0.0391 R² = 0.9973
0 0
5
10
15
20
25
30
35
t
= V cellobiose a. V t t = cellobiose + V buffer buffer solution + V enzyme enzyme = 5 mL + 44 mL + 1 mL = 50 mL Plotting [S] vs time yields an equation with a slope of m = 0.033 (mumol/mL-min). therefore, the activity is V t t * * m = (50 mL)(.033) = 1.65 mumol/min
V t t * * m
1.65 mumol/min
1 mL of enzyme 1.65 units/mL of enzyme = 165 units/mg protein
=
b. Initial reaction rate is the slope of the equation. Thus, reaction rate is m=0.033 mumol/mL-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 (mol/L) 0.0032
Initial Reaction Rate (mol/L min)
0.0049
0.148
0.0062
0.143
0.0080
0.166
0.0095
0.200
0.111
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
