Substrate recognition mechanism of prolyl aminopeptidase from Serratia marcescens.

Abstract:

:Molecular cloning of the gene and the crystal structure of the prolyl aminopeptidase [EC 3.4.11.5] from Serratia marcescens have been studied by us [J. Biochem. 122, 601-605 (1997); ibid. 126, 559-565 (1999)]. Through these studies, Phe139, Tyr149, Glu204, and Arg136 were estimated to be concerned with substrate recognition. To elucidate the details of the mechanism for the substrate specificity, the site-directed mutagenesis method was applied. The F139A mutant showed an 80-fold decrease in catalytic efficiency (k(cat)/K(m)), but the Y149A mutant did not show a significant change in catalytic efficiency. The catalytic efficiency of the E204Q mutant was about 4% of that of the wild type. The peptidase activity of the mutant (R136A) was markedly decreased, however, arylamidase activity with Pyr-bNA was retained as in the wild-enzyme. From these results, it was clarified that the pyrrolidine ring and the amino group of proline at the S1 site were recognized by Phe139 and Glu204, respectively. P1' of a substrate was recognized by Arg136. On the other hand, the enzyme had two cysteine residues. Mutants C74A and C271A were inhibited by PCMB, but the double mutated enzyme (C74/271A) was resistant to it.

journal_name

J Biochem

journal_title

Journal of biochemistry

authors

Ito K,Inoue T,Kabashima T,Kanada N,Huang HS,Ma X,Azmi N,Azab E,Yoshimoto T

doi

10.1093/oxfordjournals.jbchem.a022800

keywords:

subject

Has Abstract

pub_date

2000-10-01 00:00:00

pages

673-8

issue

4

eissn

0021-924X

issn

1756-2651

journal_volume

128

pub_type

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