Imagine that a single mutation to chymotrypsin destroys the specificity pocket by introducing a lare R-group that blocks off the opening to the pocket, so that no part of the substratecan enter the pocket. Think about what we know about what the specificity pocket does. If the mutation has only one of the impacts listed below,which one of the following is it, most likely, based on our coverage? Greately increases Kcat. Greately decreases Km. Greately increases Km. Greately decreases Kcat.

In a-c below, will the residue on the right-hand side increase or decrease the pKa of the residue on the left-hand side? Explain.

Explain why each of the criteria listed below are important in selection of a guide RNA (hint: look under Instructions on CHOPCHOP).  Under what conditions do these criteria decrease the efficiency of binding of guide RNA to its target sequence (e.g. does more, or less, self-complementarity decrease efficiency)? a) % GC (= # of G and C’s in the guide RNA sequence divided by 20 – the length of the guide RNA; this may also be called GC content) b) Self-complementarity: c) Off-Targets (0, 1, 2, 3). In your answer, explain what the numbers 0, 1, 2, 3 indicate.  For example - Is it better to have off-target sites or no off-target sites? Are off-targets in the 1 column more or less likely to decrease efficiency than off-targets in the 3 column?

Consider the mechanism of the enzyme RNase:   What would happen to the Km (i.e., would it increase, decrease, or stay the same) if the his12 was mutated to a lysine? Explain.   What would happen to the Kcat (i.e., would it increase, decrease, or stay the same) if the his12 was mutated to a valine? Explain.