STUDIES OF THE CATALYTIC MECHANISM OF AN ACTIVE-SITE MUTANT (Y14F) OF DELTA-5-3-KETOSTEROID ISOMERASE BY KINETIC DEUTERIUM-ISOTOPE EFFECTS

DELTA-5-3-Ketosteroid isomerase (EC 5.3.3.1) 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 4-beta-proton to the 6-beta-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 4-beta-proton by Asp-38 [Xue, L., Talalay, P., & Mildvan, A. S. (1990) Biochemistry 29,7491-75001. 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 10(4.7)-fold less active catalytically than the wild-type enzyme. With [4-beta-D]androst-5-ene-3,17-dione as a substrate in H2O, a lag in product formation is observed which approaches, by a first-order process, the rate observed with protonated substrate. With the protonated substrate in D2O, a burst in product formation is detected by derivative analysis of the kinetic data which approaches the rate observed with the 4-beta-deuterated substrate in D2O. The absence of such lags or bursts with the protonated substrate in H2O or with the 4-beta-deuterated substrate in D2O, as well as the detection of buffer catalysis by phosphate at pH 6.8, indicates that one or more intermediates dissociate from the enzyme and partition to substrate 31.6 times faster than to product. When corrected for these exchange effects, the k(cat) and k(cat)/K(m) values both show a primary kinetic isotope effect of 2.4 +/- 0.2 for the 4-beta-D substrate. The detection of a secondary kinetic isotope effect on k(cat)/K(m) of 1.06 +/- 0.02 with the 4-alpha-D substrate and the absence of an inverse secondary kinetic isotope effect with the 6-D substrate (1.02 +/- 0.02) indicate that enolization is rate limiting for the Y14F mutant. The primary kinetic isotope effects on k(cat)/K(m) with the 4-beta-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 4-beta-D substrate, establishing a stepwise enolization mechanism. A minimal mechanism of the reaction catalyzed by the Y14F mutation thus involves the initial stereoselective removal of the 4-beta-proton by Asp-38 to form the dienolate carbanion intermediate, which dissociates from the enzyme and is protonated in solution either at C-4 to regenerate the substrate or more slowly on the C-3 oxyanion to form the dienol, which reketonizes rapidly to form the product. Comparison of the k(off) of the intermediate from the Y14F mutant with that found with the D38N mutant indicates that the phenolic hydroxyl group of Tyr-14 contributes at least 7.6 Kcal/mol to the free energy of binding of the intermediate. Tyr-14 thus appears to play a major role not only in the formation of the dienolic intermediate but also in binding it tightly to the enzyme. 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.