Significance of the Conserved Tyr352 and Asp380 Residues in the Catalytic Activity of Bacillus stearothermophilus Aminopeptidase II as Evaluated by Site-directed Mutagenesis |
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Authors: | Long-Liu Lin Yi-Pu Chen Jia-Ci Yang Yu-Wen Hua Wen-Ching Wang Lih-Ying Kuo |
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Affiliation: | Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi 60083, Taiwan. llin@mail.ncyu.edu.tw |
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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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Keywords: | Aminopeptidase Bacillus stearothermophilus Site-directed mutagenesis Tryptophan emission fluorescence Circular dichroism |
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