Kinetic and Mechanistic Studies of Prolyl Oligopeptidase from the Hyperthermophile Pyrococcus furiosus

Abstract

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 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.Pyrococcus furiosus is a hyperthermophilic member of the domain Archaea, one of the three major phylogenetic divisions of life. There is a paucity of data on the mechanisms of homologous enzymes from the three domains (Archaea, Bacteria, and Eukarya). Such data have the potential to enhance our understanding of the evolutionary history of proteins and organisms and will certainly contribute to understanding of enzyme function at high temperatures.Certain hyperthermostable enzymes share great similarities in catalytic mechanism with their counterparts from mesophilic organisms. The ␤-glucosidase of P. furiosus has been compared with its homologue from the mesophilic bacterium Agrobacterium faecalis (1). These studies suggest that the transition states of these enzymes are extremely similar, indicating that the mechanism is highly conserved. Mechanistic and structural studies of the ␤-glucosidase (2) and glutamate dehydrogenase (3, 4) from P. furiosus have demonstrated the similarities between enzyme homologues from organisms with very different temperature optima, and s...

Metallo‐ROS in Alzheimer's Disease: Oxidation of Neurotransmitters by CuII‐β‐Amyloid and Neuropathology of the Disease

“…The rate constant k cat for dopamine oxidation by CuAb 1-20 is largely affected by the soluble form of SDS (4.5 times) and noticeably influenced (80 %) by micelles with a critical micelle concentration (CMC) of around 8 mm [37] ( Figure 2 a). This pattern fits well to a binding mode with two different binding sites, [38] whereas that of CuAb 1-40 is only slightly influenced by soluble SDS (2-fold) and greatly affected by micellar SDS (8-fold). The rate constant for the shortest CuAb 1-16 is only slightly enhanced (85 %) at a saturating amount of SDS and is 14 % enhanced by micelles.…”

Prolyl endopeptidases

“…It is stable at 4 °C and 37 °C for 24 h, and has been shown to maintain activity overnight at 85 °C [15,17]. Interestingly, P furiosus PEP is homologous to both FM PEP (30% identity, 52% similarity) and the mammalian porcine PEP (32% identity, 54% similarity) [33], even though the latter two enzymes have vastly different optimum temperatures than the P. furiosus PEP [16]. The thermal stability of FM PEP has been extensively investigated as a prototype for assessing the potential of engineering a thermostable PEP.…”

“…The P. furiosus PEP demonstrated the ability to cleave azocasein as well as the ability to hydrolyze itself [15]. These observations led to the hypothesis that substrates access the active site via a gap between the two domains which can be enlarged by the 'hinge' connecting the domains [16]. More recent experiments have demonstrated that P. furiosus PEP does not display significant activity against azocasein and that the autolysis is likely an experimental artifact [17].…”

Structure and Catalysis of Acylaminoacyl Peptidase

“…However, several papers have indicated that some serine oligopeptidases hydrolyze large proteins. For example, fibroblast activation protein has gelatinase and collagenase activities (51), oligopeptidase B cleaves at a restricted site of histone proteins (52), and POP digests itself (53,54) or degrades a splice variant protein of p40-phox (55). In all of these cases the hydrolytic reactions require unusually long times, at least overnight.…”