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.
Abstract
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 mol–1K–1
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.