Problem 1

Determine the values of Km and Vmax for the decarboxylation

of β-keto acid given the following data.

[S] in mol/l V in mM/ min

2.500 0.588
1.000 0.500
0.714 0.417
0.526 0.370
0.250 0.256

remember this equation

Problem 2
Determine the values of Km and Vmax for the reaction of carbon dioxide
and water to produce bicarbonate and hydrogen ion catalyzed by
carbonic anhydrase

[S] in mM/l 1/V in M/ sec

1.25 36000
2.50 20000
5.00 12000
20.00 6000
Problem 3
For the following aspartate reaction in the presence of inhibitor, Km = 0.00065 M.
Determine Vmax in both reactions and in the reaction without inhibitor, the Km.
Identify whether the inhibition is competitive, noncompetitive or uncompetitive.
Explain briefly -
1. how I and S bind to the E as shown by the Lineweaver Burk plot.
2. the significance of the following obtained values for:
a. Vmax
b. Km inhibitor
3. differences in:
a. slope (m)
b. x-intercept

Answers:
Problem 1
slope (m) 0.615
y-intercept (b) 1.47
Vmax 0.681 mM/min
Km 0.422 M
Problem 2
slope (m) 40000
y-intercept (b) 4000
Vmax 0.00025 mM/sec
Km 10m mM
Problem 3
Reactions Vmax Km
without inhibitor 0.2 mM/ min 0.00033 M
with inhibitor 0.2 mM/ min 0.00065 M
type competitive
 x y 4.500
0.400 1.701
4.000
1.000 2.000
1.401 2.398 3.500
1.901 2.703 3.000
4.000 3.906
2.500
slope (m) = 0.617
y-intercept (b) = 1.470 2.000

1.500

1.000
 Lineweaver-Burk equation:
y= m x + b 0.500
1 = Km x 1 + 1 0.000
V Vmax [S] Vmax 0.000 0.500 1.000 1.500 2.000

Solution:
= 1/y-intercept = slope*vmax
= 1/1.47 = 0.617*0.680
Vmax = 0.680 mM/min Km = 0.420 M

x y
4.00E+04
0.800 3.60E+04
0.400 2.00E+04 3.50E+04
0.200 1.20E+04
3.00E+04
0.050 6.00E+03
slope (m) = 40000 2.50E+04
y-intercept (b) = 4000
2.00E+04

1.50E+04
Lineweaver-Burk equation:
1.00E+04
y= m x + b
1 = Km x 1 + 1 5.00E+03
V Vmax [S] Vmax
0.00E+00
0.000 0.100 0.200 0.300 0.400
 1.00E+04

5.00E+03

0.00E+00
0.000 0.100 0.200 0.300 0.400
Solution:
= 1/y-intercept = slope*vmax
= 1/4000 = 40000*0.00025
Vmax = 0.00025 mM/sec Km = 10m mM

= 0.00065 M.
tor, the Km.
competitive.

Vmax Km From LWB plot, assuming value

without inhibitor 0.2 mM/min 0.00033 M (b) y-intercept (in both reactions)
with inhibitor 0.2 mM/min 0.00065 M x-intercept (purple)
type competitive
y=bx+a
Lineweaver-Burk equation:
y= m x + b
1 = Km x 1 + 1
V Vmax [S] Vmax

Solution: without inhibitor (purple) with inhibitor (red)

= 1/y-intercept = 1/y-intercept
= 1/5 = 1/5
Vmax = 0.2 mM/min Vmax = 0.2 mM/min

graphically Km is x-intercept (not shown in the plot)

x-intercept = -1  remember this equation
Km
Solution:
= -1/x-intercept
= -1/-3000
Km = 0.00033 M
Short answer:
1. l and s bind to the same site on E, shown by the plot where the two reactions intersects at y, theref
2.
a. Vmax is related to the turnover number of catalytic constant
b. Km is the inverse measure of the affinity of the enzyme for the substrate
3.
a. slope is the Km/Vmax
b. x-intercept is the -1/Km
 Lineweaver Burk Plot:

1.000 1.500 2.000 2.500 3.000 3.500 4.000 4.500

Lineweaver Burk Plot:

00 0.200 0.300 0.400 0.500 0.600 0.700 0.800 0.900

00 0.200 0.300 0.400 0.500 0.600 0.700 0.800 0.900

WB plot, assuming values for:

tercept (in both reactions) 5
cept (purple) -3000

his equation
ons intersects at y, therefore vmax is unchanged