Hwang Guang-Yuh, Kuo Lih-Ying, Tsai Ming-Ru, Yang Shin-Ling, Lin Long-Liu
Department of Biology, Tung-Hai University, 181 Taichung-Kan Road, Taichung, Taiwan.
Antonie Van Leeuwenhoek. 2005 May;87(4):355-9. doi: 10.1007/s10482-004-5777-z.
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.
通过定点诱变，将嗜热脂肪芽孢杆菌亮氨酸氨肽酶II（LAPII）中保守的组氨酸残基His-191、His-227、His-345和His-378替换为亮氨酸。通过镍螯合层析法纯化了过表达的野生型和突变型酶，其分子量约为44.5 kDa。在测定条件下，在H345L和H378L中未检测到LAP活性。尽管相对于野生型LAPII，H191L的Km值增加了30%以上，但该残基的改变并未导致催化效率的显著变化。H227L的Kcat/Km降低39%，部分原因是Km值增加了3.9倍。基于这些结果，表明His-345和His-378在嗜热脂肪芽孢杆菌LAPII的催化反应中起关键作用。
