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 (Pf PCP), was characterized in the context of an assembly form of the protein which is a homotetramer at neutral pH. The Pf PCP exhibited maximal catalytic activity at 90±95 8C and its activity was higher in the temperature range 30±100 8C 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 Pf PCP at alkaline and acidic pH. Using the oxidized Pf PCP and two mutant proteins (Pf C188S and Pf C142/ 188S), it was found that the peaks on the high and low temperature sides of the DSC curve of Pf PCP were produced by the forms with an intersubunit disulfide bridge between the two subunits and without the bridge, respectively, indicating the stabilization effect of intersubunit disulfide bridges. The denaturation temperature (T d ) of Pf PCP with intersubunit disulfide bridges was higher by 53 8C at pH 9.0 than that of BaPCP. An analysis of the equilibrium ultracentrifugation patterns showed that the tetrameric Pf C142/188S dissociated into dimers with decreasing pH in the acidic region and became monomer subunits at pH 2.5. The heat denaturation of Pf PCP and its two Cys mutants was highly reversible in the dimeric forms, but completely irreversible in the tetrameric form. The T d of Pf C142/188S 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.Keywords: association; calorimetry; hyperthermophile; pyrrolidone carboxyl peptidase; thermal stability.Proteins isolated from hyperthermophiles are extremely stable with denaturation temperatures near or over 100 8C. Many studies on proteins from hyperthermophiles have focused on identifying the key determinants or common factors responsible for this ultrastability. Based on recent elucidation of a large number of three-dimensional structures for hyperthermophilic proteins, trends commonly associated with elevated thermostability in the proteins are: (a) a decrease in solvent-exposed surface area Furthermore, it has been reported that oligomerization is one strategy that is used to stabilize proteins in hyperthermophilic organisms [16±21]. However, the stabilization mechanism of hyperthermophile proteins still remains to be solved.To explore the stabilization mechanism of hyperthermophile proteins, we have studied the pyrrolidone carboxyl peptidase (PCP) from a hyperthermophilic archaeon, Pyrococcus furiosus (Pf PCP), with an optimal growth temperature of 100 8C [22]. PCP (E.C. 3.4.19.3) is an enzyme that removes N-terminal l-pyroglutamic acid from peptides and proteins [23±25]. Equilibrium and kinetic studies of guanidine hydroch...

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