BCH2333A PS 5 2019

1. Suppose that the data shown below are obtained for an enzyme-catalyzed reaction
in the presence and absence of inhibitor X.
[S] (mM) v (mmol mL-1 min-1)
Without X With X
0.2 5.0 3.0
0.4 7.5 5.0
0.8 10.0 7.5
1.0 10.7 8.3
2.0 12.5 10.7
4.0 13.6 12.5
(a) Using a Lineweaver-Burke plot, determine that type of inhibition that has
occurred.
(b) Does inhibitor X combine with E, ES, or with both?
(c) Calculate KI for X assuming the final concentration of X in the reaction is 0.2 mM.
2. The enzyme cyclooxygenase (COX) is part of a larger enzyme called prostaglandin
H2 synthase. This enzyme converts the 20-carbon fatty acid arachidonic acid to lipid
messengers called prostaglandins, which play an important role in mediating
inflammation.
(a) Aspirin acetylates a specific serine residue of prostaglandin H2 synthase, preventing
arachidonate from reaching the COX active site. What kind of inhibitor is Aspirin?
(b) Ibuprofen binds noncovalently to the enzyme in its active site in the absence of
substrate. How would you expect the KM and Vmax of the enzyme to be affected by
Ibuprofen? Draw a Lineweaver-Burke plot for this enzyme in the presence and absence
of ibuprofen.
(c) How could you overcome the inhibition of the enzyme caused by ibuprofen?
 3. Three enzyme-catalyzed reactions are shown below. For each reaction, you are
given the active site and catalytic mechanism, the reaction proceeding from substrate to
product through the rate-limiting transition state, and an inhibitor of the enzyme.

For all reactions, identify the type of enzyme catalytic mechanisms used in the reaction
in the left box provided, and identify the type of irreversible inhibition for the given
inhibitor in the box on the right. For mechanisms involving acid/base catalysis, circle the
acidic/basic residue(s) directly interacting with the substrate in the active site diagram
and label as acidic/basic. Where applicable, circle the cofactor(s) or coenzyme(s) and
label each as cofactor or coenzyme.

(a)
 (b)

(c)
4. You perform an experiment to measure the initial rate of reaction for 5 nmol/mL
happydase, an enzyme that obeys Michaelis-menten kinetics. From the Lineweaver-
Burke plot, you derive a y-intercept of 0.0043 µmol-1 mL s, and generate the data found
in the table below. What is the catalytic efficiency of happydase for its substrate?

[S] (μM) vo (μmol mL-1 s-1)

320 169
160 132
80 92
40 57.2
20 32.6
10 17.5
5. You have discovered a small molecule inhibitor of happydase called testostatin. You
measure the initial velocity of the enzyme-substrate reaction in the presence of
testostatin, and you find a resulting apparent KM=52.1 μM and apparent Vmax=97.53
μmol mL-1 s-1. Indicate the mechanism of reversible inhibition of happydase by
testostatin in the box provided. Draw the Michaelis-Menten and Lineweaver-Burke plots
on the axes provided, including a curve expected for the uninhibited reaction and a
curve expected for the inhibited reaction. For the Michaelis-Menten plot, label KM,
apparent KM, Vmax, and apparent Vmax. For the Lineweaver-Burke plot, label the x-
intercept, y-intercept, and slope in terms of KM and/or Vmax.