Studies of the catalytic mechanism of an active-site mutant (Y14F) of. Delta. sup 5 -3-ketosteroid isomerase by kinetic deuterium isotope effects

Journal Article · Tue Nov 12 04:00:00 EST 1991 · Biochemistry; (United States)

OSTI ID:5617715

Xue, Liang; Talalay, P; Mildvan, A S ^[1]

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1. Johns Hopkins Univ., Baltimore, MD (United States)

{Delta}{sup 5}-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 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. 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{sup 4.7}-fold less active catalytically than the wild-type enzyme. The primary kinetic isotope effects on k{sub cat}/K{sub m} with the 4{beta}-D substrate of 2.33 {plus minus} 0.06 found in H{sub 2}O decreases to 1.16 {plus minus} 0.08 in D{sub 2}O, and the solvent isotope effect of 7.69 {plus minus} 0.18 observed with protonated substrate decreases to 3.85 {plus minus} 0.21 with the 4{beta}-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.

• OSTI ID: 5617715
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:45; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BIOCHEMICAL REACTION KINETICS
DEUTERIUM COMPOUNDS
ENZYMES
HEAVY WATER
HYDROGEN COMPOUNDS
ISOMERASES
ISOTOPE EFFECTS
KINETICS
MICROORGANISMS
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
PROTEINS
PSEUDOMONAS
REACTION KINETICS
STEROIDS
WATER