Kinetic and mechanistic studies of prolyl oligopeptidase from the hyperthermophile Pyrococcus furiosus.
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 degrees C in both H(2)O and D(2)O. Pfu POP displayed many similarities to mammalian POPs, however the solvent isotope effect (k(0)/k(1)) 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 pK(a) 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 pK(es1) and 22 kJ/mol for pK(es2), 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(-1)K(-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.
Harris
M N
MN
Department of Chemistry and Institute for Biomolecular Science, and the Department of Biology, University of South Florida, Tampa, Florida 33620, USA.
Madura
J D
JD
Ming
L J
LJ
Harwood
V J
VJ
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D017666:Deuterium Oxide / Q000378:metabolism; D019143:Evolution, Molecular; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000085822:Prolyl Oligopeptidases; D011487:Protein Conformation; D020027:Pyrococcus furiosus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry*; D013379:Substrate Specificity; D013552:Swine; D013696:Temperature; D014867:Water / Q000378:metabolism
D014867:Water; D006859:Hydrogen; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; D017666:Deuterium Oxide