B540 C588 Review Problem Set 3
1) Draw a kinetic scheme for the following multisubstrate enzyme reactions (assume two
substrates and two products): ping pong; sequential ordered; sequential random.
2) How could you use an isotope trapping experiment to distinguish between ordered and
random binding mechanisms? In the experiment with hexokinase (please refer to the
Rose paper) why is it important that all the labeled glucose can be trapped by increasing
the concentration of ATP?
3) What information can you get from an ATP/ADP exchange reaction with a kinase in
the absence of acceptor alcohol? If you see no exchange, what are some likely
explanations?
4) What information can you get from a positional isotope exchange reaction with a
kinase in the absence of acceptor alcohol? If you see no exchange, what are some likely
explanations? Diagram what you expect to see.
5) Outline the split and mix method for preparing a peptide library. Show how you
might assay this library to determine the sequence specificity of a kinase.
6) Why is it important from a medical standpoint to be able to determine the substrates
for individual protein kinases? Outline the bump and hole strategy developed by Shokat
to probe this question.
7) For what kinds of enzymes is ping pong kinetics most likely? Give an example of this.
8) What is the role of the activation loop in protein kinases? What added element of
regulation does this loop provide for?
9) What is the likely role of Asp 166 in protein kinase A (PKA)? Comment on whether it
needs to function as a base or not? Explain your reasoning. (We will discuss this at the
review session. I want more than one opinion about this. It is a thought question, there is
no right answer.)
10) Please read the Cole paper on bisubstrate analogues inhibitors of protein kinases.
According to the paper, bisubstrate analogues should have what kind of potency based on
substrates? Taking entropy into account, is this a reasonable claim? What is the
evidence based on this inhibitor for a dissociative transition state?
11) What is the major difference between an integral and a peripheral membrane protein?
Outline the basic strategies utilized by peripheral membrane proteins for membrane
association.
12) What are the various enzymes associated with phospholipid hydrolysis? Why are
these enzymes important for cell signaling?
13) The largest class of phospholipases includes the low molecular weight secreted
enzymes, which are common constituents of snake and insect venoms. What role might
these enzymes play in the function of these venoms.
14) Draw a basic mechanism for phospholipid hydrolysis catalyzed by phospholipase A-2
(PLA2). Describe the roles of any essential functional groups and/or metal ions.
15) Draw a basic scheme for the kinetics of interfacial catalysis on a membrane surface.
What are the two possible limiting modes for interfacial catalysis on such a surface.
What mode must be used if kinetics are going to be measured? Why?
16) Draw a minimal kinetic scheme for PLA2 catalysis on the surface of a vesicle.
Describe how you would probe the reversibility of the hydrolysis step. Which relative
rate constants are important for this experiment?