Abstract
The proline iminopeptidase (PchPiPA) of the white-rot fungi Phanerochaete chrysosporium is an exopeptidase specific to catalyze hydrolysis of the N-terminal proline of peptides or proteins. Its catalytic cavity is comprised of a catalytic triad (Ser107, Asp264 and His292) and an oxyanion hole (His38, Gly39, Gly40 and Pro41). In this work, several amino acid residues involved in the catalytic cavity were selected for investigation of their influences on the catalytic activity by site-directed mutagenesis. It was shown that mutation of residues (Gly39 and Gly40) involved in oxyanion hole resulted in almost complete loss of catalytic activity largely due to changes in kcat. The other residues (Gly42 and Cys45) lined at the entrance of the active cavity also yielded a profound negative effect on the activity. Mutation of the other two residues Arg130 and Gly131 which were flanked spatially by the nucleophilic attacking active site of Ser107, caused different effects on the activity. R130Aincreased catalytic efficiency due to changes in both kcat and Km; while G131V decreased the value of kcat/Km mainly due to changes in kcat. And T111Aalso caused a negative effect on the kcat. Conclusively, these amino acid residues involved in active cavity were more susceptible to be negatively affected by mutation, suggested that the active cavity of proline iminopeptidase might evolve to be less plausible.
Highlights
Proline iminopeptidase (PiP, prolyl aminopeptidase; EC3.4.11.5) is a special exopeptidase, which catalyzes cleavage of proline from the N-terminus of peptides [1]
The results showed that these amino acid residues were more susceptible to mutation, suggesting the PchPiPA may contain a relative rigid catalytic cavity
The proline iminopeptidase (PchPiPA) from Phanerochaete chrysosporium belongs to the subfamily S33.001, and their catalytic triad (Ser107-Asp264-His292) has been confirmed with site-directed mutagenesis [7]
Summary
3.4.11.5) is a special exopeptidase, which catalyzes cleavage of proline from the N-terminus of peptides [1]. The active cavity is consisted of a catalytic triad (Asp-Ser-His), which is located at the end of a deep pocket at the interface between these two domains. Previous studies with the PiP from Serratia marcescens have focused on the substrate recognition and identified several residues involved in substrate recognition by site-directed mutagenesis and structural analysis [22,23,24]. Based on its amino acid sequence, PchPiPA belongs to the subfamily S33.001, and it is somehow different from the other two fungal PiPs which have been characterized biochemically. In order to get more insights on the catalytic mechanism, site-directed mutagenesis was performed on the amino acid residues surrounding to the catalytic triad and oxyanion hole. The results showed that these amino acid residues were more susceptible to mutation, suggesting the PchPiPA may contain a relative rigid catalytic cavity
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