Review Problem Set 2 B540-C588
1) What basic caveats need to be kept in mind for site-directed mutagenesis experiments intended to reveal the role of active site residues?
2)Draw the basic mechanism for a zinc metalloprotease. What is the role of the active site zinc and the active site glu? In many zinc proteases there is an active site tyrosine that is covalently labeled by tyr-specific reagents, with concurrent loss of enzyme activity. How might you probe the role of this residue in catalysis?
Please read the Bartlett paper on transition state analogue inhibitors of carboxypeptidase A.
3) What kinetic test can be used to tell whether the inhibitors are transition state analogues?
4) On what basis do the authors rule out the possibility of glu as an active site nucleophile?
5) Draw a notional active site for alkaline phosphatase.
Define roles for each of the residues.
Draw a mechanism for the enzyme-catalyzed reaction.
Replacement of the active site serine with alanine gives an enzyme with altered catalytic properties. Predict the effects on Km and kcat.
How would you explain low levels of residual enzyme activity?
6) Delineate the major groups of nonspecific phosphatases. List whether they use an active site nucleophile and/or metal ions.
7) To trap a phosphoenzyme intermediate, what must be true about the relative rates of the microscopic (step by step) rate constants. Draw a kinetic scheme and free energy profiles to illustrate this. Explain some methods for how you might go about doing this experiment.
8) Inorganic phosphate is a potent inhibitor of alkaline phosphatase. Under some conditions, it binds so tightly that dissociation of Pi is the rate limiting step. Draw a free energy profile for this process and comment about this phenomenon in terms of differential binding and optimization of the catalytic process. Why might this adaptation be useful? How might you speed up this enzyme (in general terms)?
9) What is a Bronsted plot? Explain what such a plot is designed to show. Graph out a notional reaction of a series of aromatic phosphate esters with a beta value of -1. What is the interpretation of this? Alkaline phosphatase shows a beta value for kcat of close to zero. What possible interpretations are there of this result? Which do you think is correct?
10) How would you use stereochemistry to probe the possibility of a phosphoenzyme intermediate in the mechanism of a target phosphatase?
11) What is probed using a Bronsted plot of the pKa of the nucleophile? Please refer to figure 5b of the Herschlagg review.
Please make sure you look at the review for activity-based proteomics to answer the following questions.
12) Describe the general properties of a reagent that would be useful for activity-based proteomics of a specific class of nucleophilic hydrolase (i.e. a hydrolase that proceeds through a covalent intermediate during catalysis).
Draw a notional reagent that would be useful for:
a) serine proteases
b) cysteine proteases
13) What advantages are attendant to using “click tag” approaches. Outline this process in a general way.
14) What could you use to test whether your reagent was truly attaching to active site residues. How could this rule out nonspecific attachment to a particular enzyme? How could you use this approach to discover new inhibitors?