Modification of an enzyme carboxylate residue in the inhibition of 3-oxo-Δ5-steroid isomerase by (3S)-spiro[5α-androstane-3,2'-oxirane]-17β-ol. Implications for the mechanism of action

Article abstract of DOI:10.1021/ja00329a055

The affinity label (3S)-spiro[5α-androstane-3,2'-oxirane]-17β-ol (1β) inhibits 3-oxo-Δ⁵-steroid isomerase with a stoichiometry of one molecule of inhibitor to one enzyme monomer. The trypsin digest of the inhibited enzyme shows two distinct steroid-bound peptides, both of which are derived from residues 14-45 of the enzyme. The two modified peptides (TPS₁ and TPS₂) were isolated by HPLC, and each was shown to have an ester bond linking the steroid to the peptide fragment. Hydrolysis of each of the two peptide-steroid adducts releases a different steroid. TPS₁ gives 3α-(hydroxymethyl)-5α-androstane-3β,17β-diol (S₁) whereas TPS₂ releases 3β-(hydroxymethyl)-5α-androstane-3α,17β-diol (S₂). Tracer studies with ¹⁸O-labeled 1β show the carboxylate residue(s) involved in the ester linkages to be located on the α-face of the bound steroid. Implications of this result for the catalytic mechanism are discussed.