Catalytic mechanism of an active-site mutant (D38N) of delta 5-3-ketosteroid isomerase. Direct spectroscopic evidence for dienol intermediates.

The delta 5-3-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 4 beta-proton to the 6 beta-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 4 beta-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 10(6.0)-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 alpha,beta-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 4 beta-proton and shows primary kinetic isotope effects on enolization when either 4 alpha or 4 beta 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.(ABSTRACT TRUNCATED AT 250 WORDS)