Catalytic Mechanism of an Active-Site Mutant (D38N) of Δ5-3-Ketosteroid Isomerase. Direct Spectroscopic Evidence for Dienol Intermediates

Liang Xue, Athan Kuliopulos, Albert S. Mildvan, Paul Talalay

Research output: Contribution to journalArticlepeer-review

Abstract

The Δ5-3-ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the conversion of androst-5-ene-3,17-dione to androst-4-ene-3,17-dione by a stereospecific transfer of the 4β-proton to the 6β-position. The reaction involves two steps: (a) a rate-limiting concerted enolization, comprising protonation of the 3-carbonyl oxygen by the phenolic hydroxyl group of Tyr-14 and abstraction of the 4β-proton by the carboxylate group of Asp-38, and (b) rapid reketonization of the dienol, which may or may not be concerted. The active-site mutant D38N, which lacks the base responsible for proton transfer, is about 106.0-fold less active catalytically than the wild-type enzyme. With the D38N mutant it was demonstrated spectroscopically that the enzymatic reaction involves the conversion of the substrate to both the dienol and its anion as tightly enzyme-bound intermediates, which are then converted much more slowly to the α,β-unsaturated product. In contrast to the mechanism of the wild-type enzyme, the enolization reaction promoted by the D38N mutant is not stereospecific with respect to removal of the 4β-proton and shows primary kinetic isotope effects on enolization when either 4α or 4β or both of these protons are replaced by deuterium. Kinetic isotope effects obtained with deuterated substrates, solvent, or combinations of the two indicate that, unlike in the wild-type enzyme, protonation of the carbonyl oxygen and removal of the C-4 proton are not concerted in the D38N mutant. Furthermore, the spectroscopic detection of both dienolate and dienol intermediates and the very slow rate of product formation indicate that reketonization of the intermediate(s) is likewise a stepwise process. Loss of the active-site base (Asp-38) thus profoundly alters the mechanism of the ketosteroid isomerase reaction.

Original languageEnglish (US)
Pages (from-to)4991-4997
Number of pages7
JournalBiochemistry
Volume30
Issue number20
DOIs
StatePublished - May 1 1991

ASJC Scopus subject areas

  • Biochemistry

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