Directed Mutagenesis of the Conserved Asparagine Residuesof Bacillus Stearothermophilus Leucine Aminopeptidase II

Summary To understand the structure–function relationships of Bacillus stearothermophilus leucine aminopeptidase II (LAPII), each of the four conserved asparagine residues was replaced with leucine, aspartate, and lysine respectively by site-directed mutagenesis. The over-expressed wild-type and mutant enzymes with an apparent molecular mass of approximately 44.5 kDa were purified to homogeneity by nickel-chelate chromatography. Substitution of Asn-245, Asn-335, and Asn-341 with Lys generated variants with a dramatic loss of LAP activity. Kinetic analysis of Asn-373 variants with p-leucine-nitroanilide as the substrate revealed an increase in k_cat with no significant change in K_m, leading to a more than 2-fold increase in the catalytic efficiency. Thermostability assays showed that replacement of Asn-335, Asn-341, and Asn-373 by aspartic acid markedly increased the half-life of the enzyme at 70 °C, indicating that the deamination of these residues may have a deleterious effect on LAPII.