Enzyme-catalyzed condensation reactions which initiate the rapid peptic cleavage of substrates. 1. How the structure of an activating peptide determines its efficiency

• Published in: Biochemistry (Easton) 1981-05, Vol.20 (11), p.3177-3182
• Main Authors: Silver, Marc S, James, Susan L. T
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Enzyme Activation; Hydrogen-Ion Concentration; Kinetics; Mathematics; Oligopeptides - pharmacology; Pepsin A - metabolism; Structure-Activity Relationship; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00514a029

The addition of a small peptide can significantly increase the rate at which pepsin cleaves a substrate at pH 4.5. Why? In order to find out, we have determined spectrophotometrically the relative ability of over a dozen peptides to speed the initial rate of disappearance of Phe-Trp-NH2 and Leu-Trp-Met-Arg. Here are some of the criteria which establish the reliability of the acquired kinetic data: (1) rates depend linearly on [E] and , to a good approximation, on [activator], (2) measurements with both substrates yield the same ranking for the activators tested; (3) high-pressure liquid chromatographic investigations independently confirm conclusions derived from the spectrophotometric studies. The best activators found were Z-Ala-Phe and Ala-Leu. At 3.2 mM they are respectively 60 and 30 times more effective than an equal concentration of A-(Ala)2. The two-step mechanism given below (for Phe-Trp-NH2) best explains the structural specificity found, as well as other observations on the nature of these activated cleavages. It assumes that reaction commences when pepsin catalyzes synthesis of a peptide bond between activator and substrate. The polypeptide so formed subsequently undergoes scission at a different bond. The modified activator liberated, here designated Z-AA2-AA1-Phe, can eventually provide a variety of reaction products, as the succeeding paper demonstrates.