Amino Acid Sequence around the Epoxide-reactive Residues in Pepsin

Abstract

Abstract The amino acid sequences around two sites reactive with the specific pepsin inactivator, 1,2-epoxy-3-(p-nitrophenoxy)propane, were determined to be: Sequence I, Phe-Glu-Gly-Met-Asp-Val-Pro-Thr-Ser-Ser-Gly-Glu-Leu; Sequence II, Ile-Val-Asp-Thr-Gly-Ser-Ser-Asn. Epoxide-modified pepsin was digested separately by proteases and the epoxide-modified peptides were purified by Sephadex column chromatography and high voltage electrophoresis. When the sequences of the peptides were determined, they fell into one of the two groups shown above—Sequence I or Sequence II. The modification in Sequence I, occurring at the methionyl residue, represented an alkylation by the epoxide to form a sulfonium salt. This methionine residue is located 38 residues from the COOH terminus of pepsin. The modification of the β-carboxyl group of the aspartyl residue in Sequence II was an esterification reaction. The location of this aspartyl residue in the primary structure of pepsin is uncertain. Extensive homology exists between the epoxide-modified aspartyl Sequence II and the previously reported diazo-reactive aspartyl sequence. The sequence confirmed data from a previous study (Tang, J. (1971) J. Biol. Chem. 246, 4510) which indicated that 1,2-epoxy-3-(p-nitrophenoxy)propane reacts with two main sites of pepsin and that these sites differ from those reactive with diazo and p-bromophenacyl bromide inactivators of pepsin.