Prolyl oligopeptidase, a representative of a new family of serine proteases, is remarkably sensitive to ionic strength and has two catalytically active forms, which interconvert with changing pH [Polgár, L. (1991) Eur. J. Biochem. 197, 441-447]. To reveal whether conformational changes are associated with these effects, prolyl oligopeptidase was digested with trypsin. SDS gel electrophoresis studies demonstrated that tryptic digestion of the 75-kDa native protein generated two fragments, one having a molecular mass of 51 kDa and the other of 26 kDa. The digestion was markedly dependent on the ionic strength. Specifically, the digestion proceeded more rapidly in 0.05 M Hepes buffer than in 0.05 M Hepes buffer containing 0.5 M NaCl. Moreover, the nicked enzyme formed at low ionic strength was not stable but degraded and inactivated during an extended incubation. The digestion experiments suggested that alteration in the ionic strength elicits conformational changes in native prolyl oligopeptidase, and this may account for the enhanced catalytic activity observed at higher ionic strength. The two fragments of the nicked prolyl oligopeptidase did not separate during size-exclusion chromatography under nondenaturing conditions on a Superose 12 column and eluted in place of the native enzyme, indicating that they were strongly associated. The reactive serine residues of the nicked enzyme was labeled with tritiated diisopropyl phosphofluoridate, and the fragments were separated by size-exclusion chromatography in urea. The radioactive label was found in the large fragment. N-terminal sequencing showed that the small fragment, as well as native prolyl oligopeptidase, did not have a free N-terminal amino group, while the large fragment displayed an N-terminal sequence corresponding to residues 197-205 of native prolyl oligopeptidase. The residues around the cleaved bond may represent a linkage between two unknown domains of prolyl oligopeptidase. The nicked protease had a specificity rate constant slightly more active than the native enzyme (1.3-fold) at pH 8.0 where the high-pH form of the enzyme exists. However, at pH 6.0 where the low-pH form is predominant, the increase in the rate constant was 3-4-fold with the substrates Z-Gly-Pro-2-naphthylamide and Z-Lys-Pro-2-(4-methoxy)naphthylamide. As a consequence of the tryptic cleavage, the increase in was even higher, in particular with Z-Lys-Pro-2-(4-methoxy)naphthylamide at pH 6, where the increase was more than 1 order of magnitude. These results are consistent with the existence of two catalytically active enzyme forms.
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