Significance of the Conserved Tyr352 and Asp380 Residues in the Catalytic Activity of Bacillus Stearothermophilus Aminopeptidase II As Evaluated by Site-directed Mutagenesis

Overview

Journal Protein J

Publisher Springer

Specialty Biochemistry

Date 2008 Feb 21

PMID 18286359

Citations 2

Authors

Long-Liu Lin

Yi-Pu Chen

Jia-Ci Yang

Yu-Wen Hua

Wen-Ching Wang

Lih-Ying Kuo

Affiliations

Soon will be listed here.

Abstract

The importance of the conserved Tyr352 and Asp380 residues of Bacillus stearothermophilus aminopeptidase II (AP-II) was investigated by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in recombinant Escherichia coli M15 cells and the 45-kD proteins were purified from the cell-free extracts by Ni(2+)-NTA resin. The specific activity for Tyr352 and Asp380 replacements was decreased by more than 3.5-fold. Detailed analysis of the kinetic consequences in the mutant proteins revealed that the K (m) values were increased 1.9- to 2.6-fold with respect to wild-type enzyme. Catalytic efficiencies (k (cat)/K (m)) of mutant proteins were between 3.5- and 31-fold lower than the corresponding value of the wild-type enzyme. Tryptophan emission fluorescence and circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results indicate that residues Tyr352 and Asp380 are essential for the proper function of AP-II.

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