Identification of glutamate residues important for catalytic activity of Bacillus stearothermophilus leucine aminopeptidase II |
| |
Authors: | Hsin-Ling Yang Ruey-Shyang Chen Wenlung Chen Long-Liu Lin |
| |
Affiliation: | (1) Graduate Institute of Biotechnology, National Chiayi University, 300 University Road, 60083 Chiayi, Taiwan;(2) Department of Applied Chemistry, National Chiayi University, 300 University Road, 60083 Chiayi, Taiwan |
| |
Abstract: | 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. |
| |
Keywords: | Bacillus stearothermophilus ‰ ‰ Leucine aminopeptidase Glutamate Site-directed mutagenesis |
本文献已被 PubMed SpringerLink 等数据库收录! |
|