Kinetic and mechanistic studies of prolyl oligopeptidase from the hyperthermophile Pyrococcus furiosus

Prolyl oligopeptidase (POP) is widely distributed in mammals, where it is i mplicated in neuropeptide processing. It is also present in some bacteria a nd archaea, Because POP is found in mesophilic and hyperthermophilic organi sms, and is distributed among all three phylogenetic domains, studies of it s function and structure could lead to new insights about the evolution of enzyme mechanisms and thermostability, Kinetic studies were conducted on th e POP of the hyperthermophilic archaeon Pyrococcus furiosus (Pfu) 85 degree sC in both H2O and D2O. Pfu POP displayed many similarities to mammalian PO Ps, 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 s tep. 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 r evealed 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 imid azole 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 differen ce in enthalpy of 8.5 kJ/mol, These values suggest that two or three hydrog en 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 demo nstrated for POPs from these two organisms reflect the most highly conserve d characteristics of this class of serine protease, whereas the differences between these enzymes highlights the large evolutionary distance between t hem. Such fundamental information is crucial to our understanding of the fu nction of proteins at high temperature.