Thermal stability of pyrrolidone carboxyl peptidases from the hyperthermophilic archaeon, Pyrococcus furiosus

Abstract

The temperature adaptation of pyrrolidone carboxyl peptidase (PCP) from a hyperthermophile, Pyrococcus furiosus (PfPCP), was characterized in the context of an assembly form of the protein which is a homotetramer at neutral pH. The PfPCP exhibited maximal catalytic activity at 90-95°C and its activity was higher in the temperature range 30-100°C than its counterpart from the mesophilic Bacillus amyloliquefaciens (BaPCP). Thermal stability was monitored by differential scanning calorimetry (DSC). Two clearly separated peaks appeared on the DSC curves for PfPCP at alkaline and acidic pH. Using the oxidized PfPCP and two mutant proteins (PfC188S and PfC142/188S), it was found that the peaks on the high and low temperature sides of the DSC curve of PfPCP were produced by the forms with an intersubunit disulfide bridge between the two subunits and without the bridge, respectively, indicating the stabilization effect of inter-subunit disulfide bridges. The denaturation temperature (Td) of PfPCP with intersubunit disulfide bridges was higher by 53°C at pH 9.0 than that of BaPCP. An analysis of the equilibrium ultracentrifugation patterns showed that the tetrameric PfC142/188S dissociated into dimers with decreasing pH in the acidic region and became monomer subunits at pH 2.5. The heat denaturation of PfPCP and its two Cys mutants was highly reversible in the dimeric forms, but completely irreversible in the tetrameric form. The Td of PfC142/188S decreased as the enzyme became dissociated, but the monomeric form of the protein was still folded at pH 2.5, although BaPCP was completely denatured at acidic pH. These results indicate that subunit interaction plays an important role in stabilizing PCP from P. furiosus in addition to the intrinsic enhanced stability of its monomer.