Quantification of two-step proteolysis model with consecutive demasking and hydrolysis of peptide bonds using casein hydrolysis by chymotrypsin

Abstract

Enzymatic hydrolysis of peptide bonds becomes possible after removing steric obstacles shielding polypeptide sites against enzymatic attack, i.e. after demasking of these sites. In a simple two-step model, proteolysis was regarded as a two-step process with consecutive demasking and hydrolysis stages. A new analytical procedure was suggested to determine three experimental kinetic parameters: demasking rate constant kd, degree of initially masked peptide bonds m and maximum hydrolysis rate constant kh. The approach was shown on the example of the hydrolysis of summary casein by chymotrypsin (25°C, pH 7.5). Kinetic analysis includes the determination of an apparent Michaelis constant that we regard as a function of the degree of peptide bond hydrolysis. Two sets of hydrolysis rate constants were used to calculate the parameters of a two-step model. It was found that kd is lower than the hydrolysis rate constants for specific sites consisting of aromatic amino acid residues, and at least a half of peptide bonds is initially masked in casein. Using parameters of a two-step model, we calculated second-order rate constant and degrees of hydrolysis for specific peptide bonds as functions of the hydrolysis degree.