EFFECTS OF IONIC-STRENGTH ON THE CATALYSIS AND STABILITY OF PROLYL OLIGOPEPTIDASE

Prolyl oligopeptidase is the prototype of a new serine protease family , unrelated to trypsin and subtilisin. In contrast with these protease s, prolyl oligopeptidase is remarkably sensitive to ionic strength, be ing more active in the presence of high concentrations of salt. The en zyme has two catalytic forms, which interconvert with changing pH. To reveal the structural bases of these phenomena, the effects of 0.5 M N aCl on the stability of the enzyme were investigated by studying its d enaturation as a function of pH, temperature, and urea concentration. The three independent methods have unequivocally demonstrated that den aturation of the enzyme is promoted in the presence of NaCl. Furthermo re, destabilization of the low-pH form by urea is more significant tha n that of the high-pH form. Examination of the fluorescence emission s pectra of various denatured forms indicates that the enzyme is not ful ly unfolded in 8 M urea, nor at acidic pH. The tryptophan residues in the acid-denatured state are mainly buried. The results are interprete d in terms of the decay of the protective water shell at the higher io nic strength. The higher enthalpy and entropy of activation for heat d enaturation provide further evidence that a more ordered water stuctur e stabilizes the protein in the absence of salt. The biphasic kinetics obtained with denaturation by heat and urea suggest that the enzyme h as two domains of different stabilities.