Identification of glutamate residues important for catalytic activity of Bacillus stearothermophilus leucine aminopeptidase II.
Each of four conserved glutamate residues of Bacillus stearothermophilus leucine aminopeptidase II (BsLAPII) was replaced with aspartate, lysine, and leucine respectively by site-directed mutagenesis. The over-expressed wild-type and mutant enzymes were purified to homogeneity by nickel-chelate chromatography and the molecular mass of the subunit was determined to be 44.5 kDa by SDS-PAGE. The specific activity for the Glu-316 and Glu-340 mutants was completely abolished, while Glu-249 mutants showed comparable activity to that of the wild-type BsLAPII. Compared with the wild-type enzyme, the E250D and E250L mutant enzymes retained less than 18% of the enzyme activity and exhibited a dramatic decrease in the value of k (cat)/K (m). These observations indicate that Glu-250, Glu-316, and Glu-340 residues are critical for the catalytic activity of BsLAPII.
Yang
Hsin-Ling
HL
Graduate Institute of Biotechnology, National Chiayi University, 300 University Road, 60083 Chiayi, Taiwan.
Chen
Ruey-Shyang
RS
Chen
Wenlung
W
Lin
Long-Liu
LL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004591:Electrophoresis, Polyacrylamide Gel; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D018698:Glutamic Acid / Q000032:analysis*; D007931:Leucyl Aminopeptidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D016415:Sequence Alignment
D021122:Protein Subunits; D018698:Glutamic Acid; D007931:Leucyl Aminopeptidase