SINGLE PEPTIDE-BOND HYDROLYSIS RESYNTHESIS IN SQUASH INHIBITORS OF SERINE PROTEINASES .2. LIMITED PROTEOLYSIS OF CURCURBITA-MAXIMA TRYPSIN INHIBITOR-I BY PEPSIN

Porcine pepsin hydrolyzes the Leu7-Met8 (P2'-P3') peptide bond in Cucu rbita maxima trypsin inhibitor I (CMTI 1) in the pH range 2.0-4.8. The reaction proceeds to equilibrium between intact CMTI I and its cleave d form. The pH-independent value of the equilibrium constant (K(hyd)0 = 0.78) indicates that both forms of the inhibitor have similar Gibbs energies. The pH dependence of this constant shows that the peptide bo nd hydrolysis does not perturb ionization constants of any preexistent groups. The same equilibrium values can also be reached from the clea ved inhibitor side through pepsin-catalyzed resynthesis of the Leu7-Me t8 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 reac tions depend much less on pH and are within values typical for oligope ptide substrates of pepsin. The influence of the binding loop rigidity on slow proteolysis by pepsin and other proteinases is discussed.