Mechanism of inactivation of 3-oxosteroid Δ⁵-isomerase by 17βo-oxiranes

The affinity label (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2′-oxiran]-3-ol (5β) inactivates 3-oxosteroid Δ⁵-isomerase from Pseudomonas testosteroni by formation of a covalent bond between Asp-38 of the enzyme and the steroid. High-performance liquid chromatography (HPLC) analysis of tryptic digests of inactivated enzyme shows that two isomeric steroid-containing peptides are formed in a ratio of 9:1 at pH 7 (TPS₁ and TPS₂). Hydrolysis of each of these peptides produces a different steroid: TPS₁ releases 17α-(hydroxymethyl)estra-1,3,5(10),6,8-pentaene-3,17β-diol (S₁) whereas TPS₂ yields 170-(hydroxymethyl)estra-1,3,5(10),6,8-pentaene-3,17α-diol (S₂). Inactivation of the enzyme by (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2′-oxiran- ¹⁸O]-3-ol, followed by mass spectral analysis of the diacetate of the steroid released upon hydrolysis of the enzyme-inhibitor bond, reveals that TPS₁ is formed by attack of Asp-38 at the methylene carbon of the oxirane. In contrast, TPS₂ is produced by Asp-38 attack at the tertiary carbon. These results imply that inactivation occurs through concurrent S_N1 and S_N2 reactions of Asp-38 with the protonated inhibitor and that Asp-38 is located on the a face of the steroid when it is bound to the active site in the correct manner to react for both the S_N1 and S_N2 processes.