Mechanism of inactivation of 3-oxosteroid delta 5-isomerase by 17 beta-oxiranes.
The affinity label (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran]-3-ol (5 beta) inactivates 3-oxosteroid delta 5-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 17 alpha-(hydroxymethyl)estra-1,3,5(10),6,8-pentaene-3,17 beta-diol (S1) whereas TPS2 yields 17 beta-(hydroxymethyl)estra-1,3,5(10),6,8-pentaene-3,17 alpha-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 alpha 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.
D004852:Epoxy Compounds / Q000138:chemical synthesis* / Q000494:pharmacology; D004963:Estrenes / Q000138:chemical synthesis* / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000138:chemical synthesis*; D007202:Indicators and Reagents; D007535:Isomerases / Q000037:antagonists & inhibitors*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology; D013251:Steroid Isomerases / Q000037:antagonists & inhibitors*; D013329:Structure-Activity Relationship; D014357:Trypsin
D004852:Epoxy Compounds; D004963:Estrenes; D004988:Ethers, Cyclic; D007202:Indicators and Reagents; D010446:Peptide Fragments; D014357:Trypsin; D007535:Isomerases; D013251:Steroid Isomerases; C022660:steroid delta-isomerase