Enzyme Structure and Catalysis (5E) Practice Test

A soluble human hydrolase (E) catalyzes cleavage of an ester substrate (S) in buffered aqueous solution at 37°C. Initial rates were measured at varying $S$ with and without 10 µM inhibitor X. The data are summarized: without X, $V_{\max}=120$ nM·s$^{-1}$ and $K_m=15$ µM; with X, $V_{\max}=118$ nM·s$^{-1}$ and apparent $K_m=60$ µM. Assume enzyme concentration is unchanged and product inhibition is negligible. Based on the vignette, which outcome is most consistent with the presence of a competitive inhibitor?

Constants: none needed beyond values provided.

A soluble enzyme contains a flexible loop that closes over the active site upon substrate binding, excluding bulk water. A mutation that increases loop rigidity (without changing the identity of active-site residues) is introduced. Kinetic measurements show a decrease in $k_{cat}$ with minimal change in $K_m$. Which structural change would most likely increase enzyme activity relative to the rigid-loop mutant?