PROBLEM:

The table gives enzyme-catalyzed reaction rates (initial rate, V0) measured at various
substrate concentrations in solutions with [E] = 1.2 x 10-4 mmol/L.

1. Use the data from Experiment 1 to calculate Vmax, Km, and kcat for this enzyme-catalyzed
reaction.
2.Use the data from Experiment 2 to determine the apparent Vmax and Km in the presence of the
inhibitor.
3.From this information, determine the type of inhibition (competitive, noncompetitive or
uncompetitive) and calculate the dissociation constant KI for the inhibitor.
Experiment 1:
•Vmax is the inverse of the (1/V0) -intercept on the Lineweaver-Burke plot:

Vmax = 1/(4.6155 [L-min/mmol S]) = 0.217 mmol S/L-min

•The slope of the Lineweaver-Burke plot is equal to Km/Vmax, so Km = slope x Vmax:

Km = (0.378 [L-min/mmol S]/[L/mmol S]) x (0.217 [mmol S/L-min]) = 0.0820 mmol

S/L

•Vmax = kcat x [E]total so kcat = Vmax/[E]total

kcat = (0.217 [mmol S/L-min]/(1.2 x 10-4 [mmol E/L] = 1.81 x 103 [(mmol S/mmol
E)/min] x (1 min/60 sec)

kcat = 30.1 sec-1

Experiment 2:
Using the same methods as in experiment 1,
•Vmax(apparent) = 0.183 mmol S/L.min
•Km(apparent) = 0.152 mmol S/L
KI = Km[I]/(Kmapp - Km)
KI = (0.0820 [mmol S/L] x 0.033 [mmol I/L])/
(0.152 [mmol S/L] - 0.0820 [mmol S/L])
KI = 0.039 mM
Compare KI with Km (not Kmapp).
The inhibitor binds about twice as tightly as
the substrate.
Inhibition is competitive, but note that
with realistic data like this, there is some
uncertainty in this conclusion.
inhibition is competitive because the
inhibitor raises Km by almost 85%
lowers Vmax by only 16%.
Given the poor data (look at the
R2 values), the change is Vmax is
probably not significant.
ENZYME KINETICS – SAMPLE PROBLEM An enzymatic
assay was carried under two different sets of conditions
out using a pure substrate S. The results are tabulated
below. [S]/ Vo 10-5 M Condition A Condition B 1.5 0.21
0.08 2.0 0.25 0.1 3.0 0.28 0.12 4.0 0.33 0.13 8.0 0.44 0.16
16.0 0.40 0.18 a. Plot the data using the Lineweaver-
Burke plot b. Calculate the values of V max and K m for
both sets of conditions c. Suggest possible reasons why
the two sets of results might be different. ENZYME KINETICS
– SAMPLE PROBLEM An enzymatic assay was carried under two
different sets of conditions out using a pure substrate S. The
results are tabulated below. [S]/ Vo 10-5 M Condition A
Condition B 1.5 0.21 0.08 2.0 0.25 0.1 3.0 0.28 0.12 4.0 0.33
0.13 8.0 0.44 0.16 16.0 0.40 0.18 a. Plot the data using the
Lineweaver-Burke plot b. Calculate the values of V max and K m
for both sets of conditions c. Suggest possible reasons why the
two sets of results might be different.