Kinetic and Mechanistic Studies of Prolyl Oligopeptidase from the Hyperthermophile  Pyrococcus furiosus
Journal of Biological Chemistry 2001, 276, 1931019317

Michael N. Harris1, Jeffry D. Madura3, Li-June Ming*1, Valerie J. Harwood*2
1 Department of Chemistry and Institute for Biomolecular Science, University of South Florida, 4202 East Fowler Avenue, Tampa, FL 33620
2 Department of Biology, University of South Florida, 4202 East Fowler Avenue, Tampa, FL 33620
3 Department of Chemistry and Biochemistry, Duquesne University, 600 Forbes Avenue, Pittsburgh, PA 15282-1530

 This work was supported by NIH AREA grant R15 GM55902-02 to V.J. Harwood.

The catalytic mechanism of the prolyl oligopeptidase (POP) from the hyperthermophilic archaeon Pyrococcus furiosus (Pfu) was investigated in both H2O and D2O toward the cleavage of Z-Ala-Pro-pNA and Z-Gly-Pro-pNA. The pH and pD rate profiles display biphasic nature in which the double-deprotonated ES form is catalytically more active than the mono-deprotonated HES form. The overall solvent isotope effect (v0/v1) was ~2.0 under the conditions tested, indicating that a general base/acid-catalysis is the rate limiting step for both the mono-deprotonated and double-deprotonated forms. The pH-rate profiles were fitted by nonlinear regression analysis for a three deprotonation process, which displayed pKas of 4.25 ± 0.31, 7.15 ± 0.13, and 9.07 ± 0.12, while the temperature dependence of the pKa values revealed a DHioniz of ~4.7 kJ/mol for pKes1 and ~23 kJ/mol for pKes2. A limited analysis of substrate specificity revealed that, like other POPs,  Pfu POP is capable of cleaving a substrate with alanine at the P1 site at a relatively slow rate, and that the amino acid at the P2 site affects the rate of substrate cleavage. Like the porcine POP, and in contrast to chymotrypsin, the initial rate of Pfu POP increases hyperbolically with increasing anion concentrations. Anions affect the Michaelis-Menten parameters by acting as a nonessential activator by changing Km, but not kcat. Temperature dependence of Pfu POP was performed at two different pHs which represent the two plateaus in the pH-rate profiles. The data show large negative entropy values of ~119 and ~143 J mol1K1 at pH 6.0 and 7.6, respectively, and an enthalpy difference of 8.5 kJ/mol between the two pHs. The data suggests that hydrogen bonds may be involved during the transitional state of both active enzyme forms. A model has been constructed for Pfu POP based on crystal structure of porcine POP via sequence alignment, and is used for the discussion of the POP mechanism.

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