Single peptide bond hydrolysis/resynthesis in squash inhibitors of serine proteinases. 2. Limited proteolysis of Curcurbita maxima trypsin inhibitor I by pepsin

• Published in: Biochemistry (Easton) Vol. 33; no. 1; pp. 208 - 213
• Main Authors: Otlewski, Jacek, Zbyryt, Tomasz, Dryjanski, Marek, Bulaj, Grzegorz, Wilusz, Tadeusz
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 11.01.1994
• Subjects: Amino Acid Sequence; CUCURBITA MAXIMA; HIDROLISIS; Hydrogen-Ion Concentration; HYDROLYSE; Hydrolysis; INHIBIDORES DE ENZIMAS; INHIBITEUR D'ENZYME; Kinetics; Pepsin A - metabolism; PEPSINA; PEPSINE; Plant Proteins - isolation & purification; Plant Proteins - metabolism; PROTEOLISIS; PROTEOLYSE; Serine Proteinase Inhibitors - metabolism; Substrate Specificity; TRIPSINA; Trypsin Inhibitors - metabolism; TRYPSINE
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00167a027

Porcine pepsin hydrolyses the Leu7-Met8 (P2'-P3') peptide bond in Cucurbita maxima trypsin inhibitor I (CMTII) in the pH range 2.0-4.8. The reaction proceeds to equilibrium between in tact CMTI I and its cleaved form. The pH-independent value of the equilibrium constant (K(O)hyd = 0.78) indicates that both forms of the inhibitor have similar Gibbs energies. The pH dependence of this constant shows thai the peptide bond hydrolysis does not perturb ionization constants of any preexistent groups. The same equilibrium values can also be reached from the cleaved inhibitor side through pepsin-catalyzed resynthesis of the Leu7-Met8 peptide bond. Catalytic rate constants for the forward (hydrolysis) and reverse (resynthesis) reactions are similar. Both catalytic rate constants are strongly pH dependent, approaching the highest values at pH 2.0. Michaelis constant values for hydrolysis and resynthesis reactions depend much less oil pH and are within values typical for oligopeptide substrates of pepsin. The influence of the binding loop rigidity on slow proteolysis by pepsin and other proteinases is discussed