| Abstract: | The steroidal 3 beta-oxirane (3S)-spiro5 alpha-androstane-3,2'-oxiran]-17 beta-ol (1 beta) is an active site directed irreversible inhibitor of the 3-oxo-delta 5-steroid isomerase from Pseudomonas testosteroni. Two steroid-bound peptides (TPS1 and TPS2) were isolated by high-performance liquid chromatography (HPLC) from the trypsin digest of enzyme inactivated with 1 beta. The modified tryptic peptides (residues 14-45 of the enzyme) were further digested with chymotrypsin, each giving rise to a single steroid-containing product (CPS1 and CPS2, respectively) derived from residues 31 to 45 of the enzyme. The modified chymotryptic peptides were isolated by HPLC, and the peptide-steroid ester linkage was reduced with sodium hydroxyborohydride. Amino acid analysis of the reduced peptides gave ca. 0.5 residue of homoserine and one less residue of aspartic acid than the corresponding unreduced peptides. Sequence analysis of both reduced chymotryptic peptides revealed that homoserine was located at position 8 in the peptide sequence, corresponding to residue 38 of the enzyme. The finding that the steroidal 3 beta-oxirane, like the 17 beta-oxiranes, inactivates the isomerase via esterification of aspartic acid-38 is strong evidence that this enzyme binds steroids in at least two orientations. |
