Role of amino acid residues involved in the active cavity of proline iminopeptidase in catalytic activity

Abstract

The proline iminopeptidase (PchPiPA) of the whiterot 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 k cat . 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. R130A increased catalytic efficiency due to changes in both k cat and K m ; while G131V decreased the value of k cat /K m mainly due to changes in k cat . And T111A also caused a negative effect on the k cat . 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.