The carboxylate ion in the active center of pepsin.

Modification of a single carboxylate side chain in pepsin by 1,2-epoxy-3Qnitrophenoxy)propane abolished enzymic activity. The degree of inactivation was related to incorporation of the modifier in a 1: 1 molar ratio. The essential residue was an aspartic acid which is contained in the amino acid sequence Ile-Val-Asp-Thr-Gly-Ser-Ser-Asn. The pH rate profile of enzymic inactivation suggested the pK, of the active carboxylate ion to be below pH 3. Rate constants for the reaction of the 1,2-epoxy-3-(P-nitrophenoxy)propane with water, citrate ion, and pepsin and for the reaction of propylene oxide with pepsin were determined. Evaluation of these rate constants allowed some quantitative determination of the effect of “affinity labeling” in this system and of the nucleophilic character of the reactive carboxylate ion. In the light of these experiments and information available in the literature, potential mechanisms for pepsin catalysis are discussed.