Biochemistry I – Pset06 answer key

1. (8 pts) To determine the Km and kcat of an enzyme, you measure the rate of
product formation with varying concentrations of substrate. Because you are
uncertain as to whether the enzyme concentration will affect the determination of
Km and kcat, you carry out a parallel set of reactions at a lower enzyme
concentration. In one set of reactions, ET = 10 nM. In another, ET = 2.5 nM.
You obtain the following data:
Vo, µM/sec
[S], mM ET = 10 nM ET = 2.5 nM
0.1 7.3 1.8
0.2 13.3 3.3
0.5 26.7 6.7
1.0 40.0 10.0
2.0 53.3 13.3
5.0 66.7 16.7
10.0 72.7 18.2
50.0 78.4 19.6
i) What is VMAX when the enzyme is present at 10 nM? VMAX = ~ 80
µM/sec (1 pt). What is VMAX when the enzyme is present at 2.5 nM?
VMAX = ~ 20 µM/sec (1 pt). What is kcat for this enzyme? At either
enzyme concentration, Kcat = VMAX/[ET] = 80 µM/sec/10 nM = 20
µM/sec/2.5 nM = 80 µM/sec/10 nM = 80,000 sec-1 (1 pt). Does kcat
vary with enzyme concentration? No! (1 pt), kcat is an intrinsic
feature of the enzyme and does not depend on enzyme concentration.
ii) What is the Km of the enzyme for this substrate when the enzyme is
present at 10 nM? Km is [S] at ½ VMAX or 1 mM (1 pt). What is the
Km of the enzyme for this substrate when the enzyme is present at 2.5
nM? Again, Km is [S] at ½ VMAX or 1 mM (1 pt). Does Km vary
with enzyme concentration? No (1 pt).
iii) Under what experimental conditions might the measured Km of the
enzyme for substrate vary with enzyme concentration? The Km
might appear to depend on [ET] if [ET] were significantly higher
than [S]; not enough substrate would be present to half-saturate
the enzyme if the enzyme concentration far exceeded the substrate
concentration (1 pt). How can this situation be avoided? This
situation is avoided by working under conditions in which
[S]>>[E] (1 pt).

2. [Grader: please note that students could have obtained differences in both
Km and kcat, which would have affected all answers of this problem]. (14
pts) You work for a pharmaceutical company that has successfully developed an
effective protease inhibitor against the HIV virus. The inhibitor is a compound
that contains a cyclohexane ring at one end. The drug has been effective, for
some time, at preventing virus production in patients. Unfortunately though, a
new mutant virus that is resistant to your drug has arisen. Your task has been to
isolate the mutant protease and to carry out enzyme assays of wild type and
mutant proteases (using a standard HIV protease substrate) to determine how the
mutant protease differs from the wild type. The following data are obtained using
the same enzyme concentration for both wild type and mutant protease:

[S], µM Vo, µM/sec

wild type protease Mutant protease
2 0.167 0.062
4 0.286 0.118
8 0.444 0.210
16 0.615 0.348

i) Determine Km and VMAX for both wild type and mutant proteases for the
standard substrate.
Carry out a double reciprocal plot:

VMAX can be obtained from the y-int:

wild type VMAX = 1/y-int = ~ 1 µM/sec (2 pts)
mutant VMAX = 1/y-int = ~ 1 µM/sec (2 pts)
Km is obtained from the slope: slope = Km/VMAX and
Km = slope*VMAX: wild type Km = 10 (sec)*1µM/sec = 10 µM (2 pts).
Mutant Km = 30 (sec)*1 µM/sec = 30 µM (2 pts).
[Note: some students will have rounded off Vo and obtained slightly different
Vmax & Km. This is OK.]
ii) The enzyme concentration in all assays is 10 nM. What is kcat for the
wild type protease? kcat = VMAX/[ET] = 1 µM/sec/10 nM = 100 sec-1 (1
pt). For the mutant protease? The same as for wild type, 100 sec-1 (1
pt). [Again, some students will have obtained slightly different kcats
for wild type and mutant enzymes. This is OK].
iii) Is the mutant protease altered in its Km or kcat (or both) with respect to
the standard substrate?
 The mutant protease is altered in its Km only (1 pt).
[If students obtained a different Vmax for wt and mutant, then the
answer here should be both Km and kcat are altered.]
iv) Is the mutant protease likely to have an alteration in its catalytic Asp
residue? No (1 pt). Briefly justify our answer. Because the mutant
protease has nearly Vo, µM/sec
an identical kcat to [S], µM no inhibitor + 10 nM + 10 nM
wild type, it is very Drug A Drug B
unlikely to have an 2 0.062 0.006 0.04
alteration that affects 4 0.118 0.012 0.08
the catalytic step. (2 8 0.21 0.024 0.15
pts).
16 0.348 0.046 0.26
[Again, it is OK if they say
yes , as long as their kcat was different].

3. (14 pts) Your company gets busy and develops two new compounds with the
hope that one will turn out to be an effective inhibitor against the mutant protease.
You carry out a series of enzyme assays measuring the ability of the mutant
protease to cleave a standard substrate in the presence and absence of the first two
new compounds. The data are shown to the right:

i) Do either of the drugs greatly affect the ability of the mutant protease to
carry out the cleavage reaction (kcat)? Briefly justify your answer.
The answer is best obtained from a double reciprocal plot of the data:
Here, drug 1 is drug
A and drug 2 is drug
B.

The y-int of each plot, reflecting VMAX and kcat, differs only slightly in
the presence or absence of inhibitor:
VMAX (– inh) = 1/.96 = 1 µM/sec. VMAX (+drug A)=1/.55 = 1.8 µM/sec.
VMAX (+drug B)= 1/.64 = 1.5 µM/sec. The small differences in VMAX
 observed in the presence of the inhibitors are probably not
statistically significant. Hence the mutant protease’s kcat is not
significantly affected (2 pts). Full credit even if the differences are
noted.
ii) Determine the Km of the mutant protease for its substrate in the absence
of inhibitor (3 pts), the apparent Km of the mutant protease for its
substrate in the presence of drug A (3 pts), and the apparent Km of the
mutant protease for its substrate in the presence of drug B (3 pts). The Km
of each is obtained from the slope and VMAX (Km=slope*VMAX):
Km (-inh) = 30 (sec)*1 µM/sec = 30 µM. (2 pts)
Kmapp(+drug A) = 332 (sec)*1.8 µM/sec = 598 µM. (2 pts)
Kmapp (+drug B) = 48 (sec)*1.5 µM/sec = 72 µM. (2 pts)
iii) What is the KI of drug A for the mutant protease? α, the degree by
which Km is altered, is equal to slope (+drug A)/slope (-inh) = 332/30
= 11. Then α-1 = 10 = [I]/KI. Since [I] = 10 nM, KI = 10 nM/10 = 1
nM (4 pts).
iv) What is the KI of drug B for the mutant protease? Similarly, α for drug B
is slope (+drug B)/slope (-inh) = 48/30 = 1.6. Then α-1 = 0.6 = [I]/KI.
Since [I] = 10 nM, KI = 10 nM/0.6 = 17 nM (4 pts).
v) Which drug is likely to be a more effective inhibitor of the mutant
protease? Briefly justify your answer. Drug A, with a 17-fold lower KI,
is a more potent inhibitor of the enzyme (1 pt) because a 17-fold lower
concentration of drug B is required to half saturate the enzyme with
inhibitor (1 pt).

4. The chime page associated with this problem set shows the wild type HIV
protease in a complex with the cyclohexane-containing drug. In addition, it
shows the mutant protease in a complex with the cyclohexane-containing drug.
The KI values for the binding of the original cyclohexane-containing drug to both
wild type and mutant enzymes are shown to the right. In addition, the KI values
for the binding of each of the four new drugs to the mutant enzyme are also
provided. Please answer the following questions while viewing the chime page:

i) Which residue has been altered in the mutant HIV protease? How has
it been changed? The Valine at position 82 has been changed to
Aspartic acid in the mutant (1 pt).
ii) Provide a reasonable explanation for the change in affinity of the
mutant enzyme for the original cyclohexane-containing drug. The
mutant enzyme has a lower affinity for the cyclohexane drug
because the Val 82 to Asp82 substitution abolishes a favorable van
der Waals interaction that occurs between the non-polar hexane
ring with the Val side chain of the wild type enzyme (3 pts).
iii) Which of the four new drugs would be the worst inhibitor of the
mutant enzyme? Please refer to both the KI values provided as well as
the structures of the new drugs in your answer. Drug 3 would be the
worst inhibitor because: 1) it has the lowest affinity for the mutant
 protease (2 pts); and 2) it places a negatively charged group near
the Asp side chain in the mutant protease, resulting in charge
repulsion (2 pts).
iv) Which of the four new drugs would be the best inhibitor of the mutant
enzyme? Please refer to both the KI values provided as well as the
structures of the new drugs in your answer. Drug 1 (your drug A!)
would be the best inhibitor because it has the highest affinity for
the mutant protease (2 pts); and 2) it places a positively charged
group near the Asp side chain in the mutant protease, leading to a
favorable electrostatic interaction between inhibitor and mutant
(2 pts).

5. (10 pts, 20 min)

i) Determine the native molecular weight of the protein kinase. Please

show your work.
The elution volume of a
protein depends linearly
on the log of its
molecular weight. Thus
first plot log molecular
weight versus elution
volume for your
standards. Second,
draw a line through the
points. Third,
determine the log
molecular weight of the
protein kinase based on
its elution volume of 40
mls. This is ~ 5.15. Finally, take the anti-log of 5.15. This gives the
molecular weight of the native kinase, ~140 KDa (4 pts).

ii) Determine the denatured molecular weight(s) of the polypeptides that

make up the protein
kinase. Please show
your work (4 pts).
This is done in the
same way as above.
First plot log
molecular weight of
the standards versus
distance migrated on
the gel. Second, draw
a line through the
points. Third,
determine the log molecular weight of the protein kinase bands based
on the distance they have migrated. These are ~4.9 and ~4.5. Finally,
take the anti-log of each. This gives molecular weights of ~80 KDa
and ~32 KDa (4 pts).

iii) What is the quaternary structure of the protein kinase? Since the
native molecular weight is ~140 KDa and there are at least two
subunits, the protein kinase is most likely a heterodimer of at least one
80 KDa and at least one 32 KDa subunit. But these only add up to
~110 KDa, leaving ~30 kDa unaccounted for. Since the faster
migrating band is roughly twice the abundance of the slower, the
complex most likely consists of one 80 KDa and two 30KDa subunits
(2 pts).