Studies of the catalytic mechanism of an active-site mutant (Y14F) of Δ5-3-ketosteroid isomerase by kinetic deuterium isotope effects

Δ5-3-Ketosteroid isomerase from Pseudomonas testosteroni catalyzes the conversion of androst-5-ene-3,17-dione to androst-4-ene-3,17-dione by a stereoselective transfer of the 4β-proton to the 6β-position. The rate-limiting step has been shown to be the concerted enolization of the enzyme-bound substrate comprising protonation of the 3-carbonyl oxygen by Tyr-14 and abstraction of the 4β-proton by Asp-38. Primary, secondary, solvent, and combined kinetic deuterium isotope effects have been used to investigate the mechanism of the Y14F mutant, which lacks the proton donor and is 104.7-fold less active catalytically than the wild-type enzyme. The primary kinetic isotope effects on kcat/Km with the 4β-D substrate of 2.33 ± 0.06 found in H2O decreases to 1.16 ± 0.08 in D2O, and the solvent isotope effect of 7.69 ± 0.18 observed with protonated substrate decreases to 3.85 ± 0.21 with the 4β-D substrate, establishing a stepwise enolization mechanism. A reaction coordinate free energy contour diagram is used to compare the concerted enolization mechanism catalyzed by the wild-type enzyme with the stepwise carbanion mechanism catalyzed by the Y14F mutant and the stepwise oxycarbonium ion mechanism catalyzed by the D38N mutant