20.

320 Spring 2006

Exam 2 Review Problems

1) How does Km change with increasing [I] for an competitive inhibitor? For an non-
competitive inhibitor?

2) The Briggs-Haldane treatment of a single-substrate, enzyme-catalyzed reaction is as

follows:

k1 k2 k3

E + S <----> ES <----> EP <----> E + P

k-1 k-2 k-3

a. Derive an expression for v = k3[EP]. What is Km and kcat?

b. What assumptions do we need to make in order to obtain the Michaelis-Menten
Km and kcat expressions from the ones you found in part (a)?

3) Would EC50 likely be higher for a competitive or a non-competitive inhibitor?

4) A tethered ligand binding to a tethered receptor goes through the following bulk reaction:

+
a. T/F: According to Jencks Theory, the enthalpy of the bulk binding is equal to the
sum of the enthalpy of binding at site A and the enthalpy of binding at site B.
b. T/F: According to Jencks Theory, the entropy of the bulk binding is equal to the
sum of the entropy of binding at site A and the entropy of binding at site B.

5) Under what conditions may we assume pseudo first-order approximations for ligands
binding to receptors on cell surfaces?

6) We know that a particular receptor is internalized upon ligand binding.

a. Using 125I-labeling, we observe that the number of internal R-L complexes first
increases, but eventually starts to gradually decrease for the remainder of the
experiment duration (~1 hour). List at least two “life-cycle” processes that could
explain this behavior.
b. At low ligand concentrations, we do not observe any internal R-L complexes at all.
List at least two “life-cycle” processes that could explain this behavior.
7) Given the following partial system:

KU,B
KD,A

KU,A KD,B

KD,A = 10-4
KU,B = 10-3
KD,B = 10-5

a. What is KD,AB?
b. What is KU,A?
c. What is, [A]eff, the effective concentration of A?
d. What is the ratio of the dissociation of A in the multivalent scheme above
compared to the monovalent binding situation?
e. What characteristics of the linker between A and B would increase KD,AB?
Decrease KD,AB?

8) A pesticide inhibits the activity of a particular enzyme A, which can therefore be used to
assay for the presence of the pesticide in an unknown sample.
a. In the laboratory, the initial rate data obtained are shown below. The rate, v, is
measured in units of 1x106 mol/(L-min). Is the pesticide a competitive or
noncompetitive inhibitor? Determine Ki, vmax, and Km.

[S] (mol/L) v (no inhibitor) v (10-5 M inhibitor)

3.3 x 10-4 56 37
5.0 x 10-4 71 47
6.7 x 10-4 88 61
1.65 x 10-3 129 103
2.21 x 10-3 149 125

b. After 50 mL of the same enzyme solution in part (a) is mixed with 50 mL of

8x10-4 M substrate and 25 mL of sample, the initial rate observed is 18
µmol/(min-L). Assuming that there are no other inhibitors or substrates present in
the sample, what is the pesticide concentration in the unknown sample?
9) We have a receptor on a cell surface that is produced by the cell at a rate SR. The
receptor binds with ligand at a rate kf, and the complex dissociates with a rate kr. The
ligand-receptor complex is internalized (endocytosis) at a rate kec. The free receptor is
also internalized (endocytosis) at a rate ker. The internalized receptors and complexes are
either degraded or recycled. A fraction, fxc, of the internalized complex are recycled,
while a a fraction, fxr, of the internalized free receptor are recycled. All other internalized
receptors and complexes are degraded. The internalized complex is both recycled and
degraded at a rate kxc. The internalized free receptor is both recycled and degraded at a
rate kxr. You can assume in this problem that internalized complexes do not dissociate
inside the cell.

Write out the differential equations for all relevant species (Rs, Ri, Cs, Ci) and the

conservation equations for R0 and L0 (watch your units).