Evidence for an acyl intermediate in pepsin-catalysed reactions

Sephadex G-75, G-50 and G-25F columns, and further purification has been achieved by standard electrophoretic and chromatographic procedures. The tryptic digests with diphenylcarbamoyl chloridetreated trypsin gave four of the five lysine residues in unique peptides and a considerable number of pep- tides originating from chymotryptic cleavages. The N-terminal region and overlapping sequences around some of the lysine residues were obtained from a subtilisin digest of the maleylated protein by the diagonal electrophoresis method. The sequences of the purified peptides were determined by the dansyl- Edman method (Gray, 1968). Almost all of the 310 residues of the protein have been accounted for in 30 unique peptides. These were compared with the par- tial sequences available for porcine pepsin (see Dayhoff, 1969) and for chymosin (B. Foltmann, personal communication). The comparison shows three areas with similarity in addition to the activesite peptide. These are: 17 residues at the N-terminal of the active enzymes, Ala-Ala-Ser-Gly-Val-Ala-Thr-Asn-Thr-Pro-Thr-Ala-Asn-Asx-Glx-Glx-Tyr-; the sequence -Asp-Phe-Gly-Phe-Ile-Asp-Ser-Ser-Lys-Tyr-; and 27 residues at the C-terminal end of the molecule, -Ser-Ile-Gly-Asp-Ile-Phe-Leu-Lys-Ser-Gln-Tyr-Val-Val-Phe-Asx-Ser-Asx-Gly-Pro-Glx-Leu-Gly-Phe-Ala-Pro-Gln-Ala. These findings pro- vide good evidence for the homology between peni- cillopepsin and the mammalian acid proteases.