Modulation of the Enzymic Activity of Chicken Pepsin by the Covalent Modification of Its Single ‐ SH Group

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Abstract
The single cysteinyl residue of chicken pepsin was modified with a wide spectrum of reagents to produce mixed disulfides or alkylated derivatives. All these derivatives showed enhanced catalytic activity towards the synthetic peptide Z‐Ala‐Ala‐Phe‐Phe‐OPrP, where OPrP is the 3‐(4‐pyridyl)‐propyl‐1‐oxy group. The overall catalytic constant kcat/Km for these derivatives was 4‐25‐fold larger than that of the native enzyme. The activity of the enzyme towards denatured hemoglobin was slightly decreased (10–45%) by these modifications. When the mixed disulfide derivatives were treated with excess mercaptan, the sulfhydryl group was regenerated and activity reverted to that of the native enzyme. The ‐SH group of chicken pepsin reacted preferentially with reagents containing an aromatic group. The reaction was found to depend on the ionization of a single group, presumably the ‐SH itself, with a pKa= 7.5. The rate of reaction of the fully deprotonated species with various disulfides was 100–1000‐fold smaller than that of the ‐ SH group of glutathione. It is suggested that the groups attached covalently to the sulfhydryl also interact with other amino acid side chains in the protein thereby affecting the active center of chicken pepsin.