Histidines 345 and 378 of Bacillus stearothermophilus leucine aminopeptidase II are essential for the catalytic activity of the enzyme.

The conserved histidine residues, His-191, His-227, His-345, and His-378, in Bacillus stearothermophilus leucine aminopeptidase II (LAPII) were replaced with leucine by site-directed mutagenesis. The overexpressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular masses were approximately 44.5 kDa. Under assay conditions, no LAP activity was detected in H345L and H378L. Although the Km value for H191L increased more than 30% with respect to the wild-type LAPII, alteration in this residue did not lead to a significant change on the catalytic efficiency. The 39% decrease in Kcat/Km for H227L was partly caused by a 3.9-fold increase in Km value. Based on these results, it is suggested that His-345 and His-378 play a crucial role in the catalytic reaction of B. stearothermophilus LAPII.