title: Single peptide bond hydrolysis/resynthesis in squash inhibitors of serine proteinases. 2. Limited proteolysis of Curcurbita maxima trypsin inhibitor I by pepsin.
abstract: Porcine pepsin hydrolyzes the Leu7-Met8 (P2'-P3') peptide bond in Cucurbita maxima trypsin inhibitor I (CMTI I) in the pH range 2.0-4.8. The reaction proceeds to equilibrium between intact CMTI I and its cleaved form. The pH-independent value of the equilibrium constant (Khyd0 = 0.78) indicates that both forms of the inhibitor have similar Gibbs energies. The pH dependence of this constant shows that 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 on 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.
authors.affiliation: Institute of Biochemistry, University of Wroclaw, Poland.
authors.forename: J
authors.initials: J
authors.lastname: Otlewski
authors.forename: T
authors.initials: T
authors.lastname: Zbyryt
authors.forename: M
authors.initials: M
authors.lastname: Dryjański
authors.forename: G
authors.initials: G
authors.lastname: Bulaj
authors.forename: T
authors.initials: T
authors.lastname: Wilusz
mesh_terms: D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010434:Pepsin A / Q000378:metabolism*; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000378:metabolism; D013379:Substrate Specificity; D014361:Trypsin Inhibitors / Q000378:metabolism*
chemical_list: C081870:CMTI protein, Cucurbita maxima; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D010434:Pepsin A