The Structure and Function of Acid Proteases

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Abstract
Bovine rennin [EC 3.4.23.4] was inactivated by stoichiometric reaction with: 14C-labeled diazoacetylnorleucine methyl ester (DAN) in the presence of cupric ions. The inactivated enzyme was hydrolyzed with pepsin [EC 3.4.23.1] and the digest was fractionated by column chromatography on Sephadex G-25 and high voltage paper electrophoresis. A single labeled peptide fraction thus obtained was treated with aqueous pyridine to cleave the ester linkage between the peptide and the reagent moiety and then subjected to high voltage paper electrophoresis under the same conditions as used in the first electrophoresis. A major peptide which is thought to be the DAN reactive-site peptide, was isolated and sequenced to be Asp-Thr-Gly-Thr-Ssr-Leu. Bovine rennin was also inactivated by reaction with 1, 2-epoxy-3-(P-nitrophenoxy)-propane (EPNP) with incorporation of 2 EPNP moieties per molecule. The inactivated enzyme was hydrolyzed with pepsin and the EPNP reactive-site peptides were isolated in the same way as in the case of the DAN-reactive peptide. One of the peptides thus obtained was sequenced to be Asp-Thr-Gly-Ser-Ser. These results show that bovine rennin has two distinct aspartic acid residues in the active site and that the β-carboxyl group of one of these residues reacts specifically with DAN and thatof the other with EPNP. The amino acid sequences around these aspartic acid residues in the isolated peptides are identical with the amino acid sequences around the DAN- and EPNP-reactive aspartic acid residues in pepsin. These results thus demonstrate a close homology between pepsin and rennin.

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