Studying the Active Site Pocket of Staphylococcus hyicus Lipase by Site-Directed Mutagenesis
Authors: Chang R.C.1; Chen J.C.2; Shaw J.F.2, 3
Source: Biochemical and Biophysical Research Communications, Volume 229, Number 1, December 1996 , pp. 6-10(5)
Publisher: Academic Press
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Abstract:
Site-directed mutagenesis of a previously constructed, recombinant Staphylococcus hyicus lipase (49 kDa) showed that Val363 played a role in catalysis and substrate-binding. In comparison with wild type enzyme, the 64% and 89% decrease in the catalytic efficiency (kcat/ K m ) of the V363N and V363A enzymes, respectively, were largely caused by a 3.5- and 5.5-fold increase in the substrate-binding affinity ( K m ), respectively. In comparison with wild type enzyme, a G371A enzyme showed a 40% decrease in the K m , suggesting that G371 was important for substrate-binding specificity. Site-directed mutagenesis of the active site Asp559 revealed that in comparison with wild type enzyme, a D559E enzyme exhibited a 47% decrease in the kcat/ K m but a twofold increase in the K m for p -nitrophenyl butyrate, suggesting that Asp-559, a component of the catalytic triad, was involved in substrate-specificity.
Language: English
Document Type: Research article
Affiliations: 1: Department of Sea-Food Technology, China Junior College of Marine Technology, Taipei, 111, Taiwan 2: Institute of Marine Biotechnology, National Taiwan Ocean University, Keelung, 20224, Taiwan 3: Institute of Botany, Academia Sinica, Taipei, 11529, Taiwan
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