Studies of the mechanism of the delta 5-3-ketosteroid isomerase reaction by substrate, solvent, and combined kinetic deuterium isotope effects on wild-type and mutant enzymes

Journal Article · Tue Aug 14 04:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00484a019· OSTI ID:6073273

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

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1. Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA)

delta 5-3-Ketosteroid isomerase catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by a conservative tautomeric transfer of the 4 beta-proton to the 6 beta-position using Tyr-14 as a general acid and Asp-38 as a general base. On deuteration of the 4 beta-position (97.0%) of the substrate, kcat(H)/kcat(4 beta-D) is 6.1 in H2O and 6.3 in D2O. The solvent isotope effect, kcat(H2O)/kcat(D2O), is 1.6 for both the 4 beta-H and 4 beta-D substrates. Mutation of Tyr-55 to Phe lowers kcat 4.3-fold; kcat(H)/kcat/4 beta-D is 5.3 in H2O and 5.9 in D2O, and kcat(H2O)/kcat(D2O) with the 4 beta-H and 4 beta-D substrates is 1.5 and 1.7, respectively, indicating concerted general acid-base catalysis in either the enolization or the ketonization step of both the wild-type and the Tyr-55----Phe (Y55F) mutant enzymes. An additional slow step occurs with the Y55F mutant. Smaller isotope effects on Km are used to estimate individual rate constants in the kinetic schemes of both enzymes. On deuteration of the 4 alpha-position (88.6%) of the substrate, the secondary isotope effect on kcat/Km corrected for composition is 1.11 +/- 0.02 with the wild-type enzyme and 1.12 +/- 0.02 with the Y55F mutant. These effects decrease to 1.06 +/- 0.01 and 1.07 +/- 0.01, respectively, when the 4 beta-position is also deuterated, thereby establishing these to be kinetic (rather than equilibrium) secondary isotope effects and to involve a proton-tunneling contribution. Deuteration of the 6-position of the substrate (92.0%) produces no kinetic isotope effects on kcat/Km with either the wild-type (1.00 +/- 0.01) or the Y55F mutant (1.01 +/- 0.01) enzyme.

• OSTI ID: 6073273
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:32; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BIOCHEMICAL REACTION KINETICS
CHEMICAL REACTIONS
DEUTERATION
DEUTERIUM
ENZYMES
HYDROGEN ISOTOPES
ISOMERASES
ISOTOPE APPLICATIONS
ISOTOPE EFFECTS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAGNETIC RESONANCE
MOLECULAR STRUCTURE
MUTANTS
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ODD-ODD NUCLEI
REACTION KINETICS
RESONANCE
SPECTRA
STABLE ISOTOPES
SUBSTRATES
TRACER TECHNIQUES