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Originally published In Press as doi:10.1074/jbc.M010489200 on February 20, 2001

J. Biol. Chem., Vol. 276, Issue 22, 19310-19317, June 1, 2001
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Kinetic and Mechanistic Studies of Prolyl Oligopeptidase from the Hyperthermophile Pyrococcus furiosus*

Michael N. HarrisDagger , Jeffry D. Madura§, Li-June MingDagger , and Valerie J. Harwood||**

From the Dagger  Department of Chemistry and Institute for Biomolecular Science, and the || Department of Biology, University of South Florida, Tampa, Florida 33620 and the § Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, Pennsylvania 15282-1530

Prolyl oligopeptidase (POP) is widely distributed in mammals, where it is implicated in neuropeptide processing. It is also present in some bacteria and archaea. Because POP is found in mesophilic and hyperthermophilic organisms, and is distributed among all three phylogenetic domains, studies of its function and structure could lead to new insights about the evolution of enzyme mechanisms and thermostability. Kinetic studies were conducted on the POP of the hyperthermophilic archaeon Pyrococcus furiosus (Pfu) 85 °C in both H2O and D2O. Pfu POP displayed many similarities to mammalian POPs, however the solvent isotope effect (k0/k1) was 2.2 at both high and low pH, indicating that general base/acid catalysis is the rate-limiting step. The pH-rate profiles indicated a three-deprotonation process with pKa values of 4.3, 7.2, and 9.1. The temperature dependence of these values revealed a heat of ionization of 4.7 kJ/mol for pKes1 and 22 kJ/mol for pKes2, suggesting the catalytic involvement of a carboxyl group and an imidazole group, respectively. Temperature dependence of the catalytic rate was assessed at pH 6.0 and 7.6. Entropy values of -119 and -143 Jmol-1K-1 were calculated at the respective pH values, with a corresponding difference in enthalpy of 8.5 kJ/mol. These values suggest that two or three hydrogen bonds are broken during the transition state of the acidic enzyme form, whereas only one or two are broken during the transition state of the basic enzyme form. A model has been constructed for Pfu POP based on the crystal structure of porcine POP and the sequence alignment. The similarities demonstrated for POPs from these two organisms reflect the most highly conserved characteristics of this class of serine protease, whereas the differences between these enzymes highlights the large evolutionary distance between them. Such fundamental information is crucial to our understanding of the function of proteins at high temperature.

* This work was supported by National Institutes of Health Academic Research Enhancement Award Grant R15 GM55902-02 (to V. J. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence may be addressed: Dept. of Chemistry and Institute for Biomolecular Science, University of South Florida, 4202 East Fowler Ave., Tampa, FL 33620. Tel.: 813-974-2220; Fax: 813-974-1733; E-mail: ming@chuma.cas.usf.edu.

** To whom correspondence may be addressed: Dept. of Biology, University of South Florida, 4202 East Fowler Ave., Tampa, FL 33620. Tel.: 813-974-1524; Fax: 813-974-3263; E-mail: vharwood@chuma1.cas.usf.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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L. Shan, I. I. Mathews, and C. Khosla
Structural and mechanistic analysis of two prolyl endopeptidases: Role of interdomain dynamics in catalysis and specificity
PNAS, March 8, 2005; 102(10): 3599 - 3604.
[Abstract] [Full Text] [PDF]


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