Worksheet 1 Enzyme Kinetics

1. An enzyme with a KM of 1x10-3 M was assayed using an initial substrate concentration of

3x10-5 M. After 2 min, 5 percent of the substrate was converted. How much substrate will be
converted after 10 min, 30 min, 60 min? How long must the reaction be run to achieve 99%
conversion? (Assume that the enzyme follows Michaelis-Menten kinetics.)
2. Anaerobic digestion is the degradation of complex organic matter to gaseous products, CO2
and CH4. Although the complete process involves complex interacting microbial species, the
majority of methane formed in an anaerobic digestion is produced by acetate-utilizing
methanogens. Yang and Okos have studied the kinetics of methanogenesis from acetat e for
Methanococcus mazei and Methanosarcina barkeri in batch culture. Significant substrate
inhibition was observed for both methanogens grown on acetate. Rate constants estimated from
growth data are as follows:

Strain µmax (h-1) KS(g acetate/L) KI (g acetate/L)

M. Mazei S6 0.029 1.0 48.7

Using these data, calculate for each methanogen the optimal acetate concentration and the
maximum observable specific growth rate.
Worksheet 3.

The hydration of CO2 is catalyzed by carbonic anhydrase as follows:

𝐸
𝐻2 𝑂 + 𝐶𝑂2 ↔ 𝐻𝐶𝑂3− + 𝐻 +

The following data were obtained for the forward and reverse reaction rates at pH 7.1 and
enzyme concentration of 2.8 × 10−9 M.

v is the initial reaction rate at the given substrate concentration. Calculate the forward and
reverse catalytic and Michaelis constants.
 Hydration (forward reaction)
y Linear (y) y = 40x + 4
40 R² = 1

35
30
25
1/r

20
15
10
5
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
1/s

Hydration (backward reaction)

y Linear (y)

100
90 y = 163.86x + 12.86
80 R² = 0.9997
70
60
1/r

50
40
30
20
10
0
0 0.1 0.2 0.3 0.4 0.5 0.6
1/s
Problem 4.

a.) Evaluate KM and Vmax by a Lineweaver-Burk Plot.

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

Lineweaver-Burk
y Linear (y)

10
9
y = 0.0172x + 3.6303
8 R² = 0.9156
7
6
1/r

5
4
3
2
1
0
0 50 100 150 200 250 300 350
1/Cs

slope intercept =1/rmax

rmax=1/slope intercept
rmax=1/3.6303=0.2755 mol per L min

slope =Km/rmax
Km=(rmax)(slope)=(.2755)(0.0172)=0.004786
mol/L
 b.) Using an Eadie Hofstee plot, evaluate Vmax and KM

Eadie-Hofstee
y Linear (y)

0.25

0.2 y = -0.0043x + 0.2645

R² = 0.6584

0.15
r

0.1

0.05

0
0 5 10 15 20 25 30 35 40
r/cs

y intercept=rmax=0.2645 mol per min L

slope=-Km=0.0043 mol per L

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