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

Shanta Bantia, Charles L Bevins, Ralph M. Pollack

Research output: Contribution to journalArticlepeer-review

11 Scopus citations


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 SN1 and SN2 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 SN1 and SN2 processes.

Original languageEnglish (US)
Pages (from-to)2606-2609
Number of pages4
Issue number11
StatePublished - 1985
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


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