PH-DEPENDENT MECHANISM IN THE CATALYSIS OF PROLYL ENDOPEPTIDASE FROM PIG MUSCLE

Prolyl endopeptidase, an enzyme exhibiting high specificity towards the Pro-Xaa bond, is thought to play an important role in the metabolism of biologically active peptides. (a) It has been found that pig muscle is an appropriate source for the preparation of a reasonable quantity of enzyme. Thus, 1 kg muscle yields 1-1.5 mg enzyme, homogeneous by fast protein liquid chromatography. (b) Bulky reagents reacting with cysteine residues inactivate the enzyme almost completely, whereas the small molecule iodoacetamide only partially inhibits the catalytic activity. This indicates that the thiol group is not essential for catalysis, but it is located at or near the active site. (c) Kinetic analysis has indicated that prolyl endopeptidase is remarkably sensitive to ionic strength. Addition of salts, e.g. sodium chloride, to the reaction mixture, up to 0.5 M, considerably enhances the rate of acylation, suggesting that charged groups of the protein exert significant effects on the catalytic site. (d) This is supported by the doubly sigmoidal character of the pH-rate profile. The existence of the doubly sigmoidal curve also indicates that the enzyme has two forms, which exhibit different activities and interconvert with changing pH. (e) The low-pH form displays a significant kinetic deuterium isotopic effect (1.70 and 1.89 in the absence and in the presence of 0.5 M NaCl, respectively), as usually observed in the serine protease catalysis. In contrast, the high-pH form, which is physiologically operative, however, has practically no kinetic deuterium effect (1.09 and 1.15 in the absence and in the presence of 0.5 M NaCl, respectively). It is concluded that a general base/acid-catalyzed acylation step is rate-limiting in the lower pH range, and an isotopically silent step, probably a conformational change preceding acylation, dominates the reaction in the physiological pH range.