BC 2016 Additional problems on enzyme kinetics - KEY

1. The following data were obtained from an enzyme kinetics experiment.

Graph the data using a Lineweaver-Burk plot and determine, by inspection of
the graph, the values for Km and Vmax.

[S] (µM) V (nmol/min)

_______ ___________

0.20 1.43
0.26 1.67
0.33 2.08

m
1.00 3.33

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Km=0.5 µM, Vmax= 5nmol/min

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2. Calculate the maximum rate for an enzyme if its kcat = 1.4 x 104 s-1 Km = 90
µM. Is this enzyme very efficient?
Th

Km=90×10-6M

Specificity constant (or kinetic efficiency) = kcat/Km = 1.4×104/90×10-6 =

155.5×106s-1 M-1=1.55×108M-1s-1

Yes, it is efficient because the enzyme has achieved catalytic efficiency.

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 3. The effect of an inhibitor on an enzyme was tested and the experiment gave
the results below. Plot the data and determine, by inspection of the graph,
what type of inhibition is involved.

[S] µM V (µmol/min) V (µmol/min) V (µmol/min)

with 0.0 nM with 25 nM with 50 nM
Inhibitor Inhibitor Inhibitor
______ ___________ ___________ ___________

0.4 0.22 0.21 0.20

0.67 0.29 0.26 0.24
1.00 0.32 0.30 0.28
2.00 0.40 0.36 0.33

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Uncompetitive
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4. You perform a kinetics experiment on the enzyme phosphatidylinositol

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synthase (PI synthase) using as substrate radiolabeled inositol in a tracer

amount mixed with unlabeled inositol.
Th

PI Synthase
CDP-DAG + Inositol -----------------------------à Phosphatidylinositol

Using a scintillation counter, you have determined that the inositol substrate has a
specific radioactivity of 100 dpm (disintegrations per minute) / 1 nmol of inositol.

You collect the following data that represent the amount of radiolabeled
phosphatidylinositol formed (data in dpm) after a 10 min reaction using 5 µM enzyme

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 [S] nmol/100 µl Reaction Volume dpm Recovered in Product Formed
____________________________ ____________________________

250 10,000
500 16,000
750 17,500
1,000 21,700

a) Determine nmol product formed per min for each substrate concentration used.
Velocity
[S] (mM)
(nmol/min)
2.5 10.0
5.0 16.0

m
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7.5 17.5
10.0 21.7

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b) Prepare a Lineweaver-Burk plot of the data.

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c) Determine Km and Vmax for PI synthase.

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Km=5.55mM, Vmax=33.3nmol/min

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 5. Salicylate (aspirin) inhibits the catalytic action of glutamate dehydrogenase.
Plot the data two ways: (1) v vs. [S] (2) 1/v vs. 1/[S] on graph paper. Estimate
the Vmax and Km in the presence and absence of this inhibitor.
Product per minute (microgram)
Substrate (mM) No Inhibitor 40mM Salicylate
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 0.39 0.15
16.0 0.46 0.17

Graph:

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Th

Vmax=0.5ug/min; Vmax'=0.2ug/min, Km=2.1mM

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 a) How well do the estimates agree from the two plots.
Quite well. The [S] at half Vmax in the MMK graph is ~2mM which is
similar to that obtained thru the Line-Weaverburk plot
b) From the data, can you determine the type of inhibition?
Non-competitive
c) Determine the ratio of [ES] uninhibited to [ES] inhibited at 4mM [S].
Ratio of v/Vmax(uninhibited)/v/Vmax(inhibited) = 1.01

6. Another type of inhibitor of glutamate dehydrogenase was tested.

a) Do the same calculations on this inhibitor as in Q. 6.
b) From the data, can you determine the type of inhibition?
c) How well do they agree?
Product per minute (nanomol)
Substrate (mM) No Inhibitor 6mM Inhibitor

m
2.0 139 88

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3.0 179 121

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4.0 213 149

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10.0 313 257

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15.0 370 295
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Graph: Must extrapolate by hand
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Competitive; Vmax=500nmol/min; Km=5mM, Km'=10mM

Both graphs agree quite well. Vmax =500nmol/min. At 1/2Vmax (250nmol/min) the Km
for uninhibited is ~5.5mM, Km for inhibited is ~10mM.

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