TITLE:
Role of amino acid residues involved in the active cavity of proline iminopeptidase in catalytic activity
AUTHORS:
Kangkang Xing, Hong Feng
KEYWORDS:
Proline Iminopeptidase; Site-Directed Mutagenesis; Oxyanion Hole; Active Cavity; Catalytic Kinetics; Phanerochaete chrysosporium
JOURNAL NAME:
Advances in Biological Chemistry,
Vol.3 No.3,
May
31,
2013
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.