Inactivation of Bacillus stearothermophilus leucine aminopeptidase II by hydrogen peroxide and site-directed mutagenesis of methionine residues on the enzyme.
Leucine aminopeptidases (LAPs) are exopeptidases that remove the N-terminal L-leucine from peptide substrates. Oxidative stability assay showed that the recombinant Bacillus stearothermophilus LAP II (rLAPII) was sensitive to oxidative damage by hydrogen peroxide at the elevated temperature. The H2O2-treated enzyme experienced obvious changes in the secondary structure when the oxidant concentration increased to 300 mM. To investigate the role of methionine residues on the oxidative inactivation, each of the five methionine residues in the rLAPII was replaced with leucine by site-directed mutagenesis. The mutant enzymes with an apparent Mr of approximately 44.5 kDa were overexpressed in Escherichia coli and were purified to homogeneity by nickel-chelate chromatography. The specific activities for Met82Leu, Met88Leu, Met254Leu, and Met382Leu were similar to that of the wild-type enzyme, whereas a reduced activity was observed in Met136Leu. The 50% decrease in the catalytic efficiency (kcat/Km) for Met136Leu was caused by 47% decrease in kcat value. As compared with the wild-type enzyme, all mutant proteins were more sensitive to the oxidant, implying that the methionine residues of B. stearothermophilus LAP II are important for the protection of the enzyme from oxidative inactivation.
Kuo
Lih-Ying
LY
Department of Biology, Tung-Hai University, 181 Talchung-Kan Road, Taichung, Taiwan.
Hwang
Guang-Yuh
GY
Yang
Shin-Ling
SL
Hua
Yu-Wen
YW
Chen
Wenlung
W
Lin
Long-Liu
LL
D019943:Amino Acid Substitution / Q000235:genetics; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007931:Leucyl Aminopeptidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008715:Methionine / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010084:Oxidation-Reduction
D008715:Methionine; D006861:Hydrogen Peroxide; D007931:Leucyl Aminopeptidase