Studying the active site pocket of Staphylococcus hyicus lipase by site-directed mutagenesis.
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/Km) of the V363N and V363A enzymes, respectively, were largely caused by a 3.5- and 5.5-fold increase in the substrate-binding affinity (Km), respectively. In comparison with wild type enzyme, a G371A enzyme showed a 40% decrease in the Km, 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/Km but a twofold increase in the Km for p-nitrophenyl butyrate, suggesting that Asp-559, a component of the catalytic triad, was involved in substrate-specificity.
Chang
R C
RC
Department of Sea-Food Technology, China Junior College of Marine Technology, Taipei, Taiwan.
Chen
J C
JC
Shaw
J F
JF
D001665:Binding Sites / Q000235:genetics; D002087:Butyrates / Q000378:metabolism; D007700:Kinetics; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D013210:Staphylococcus / Q000201:enzymology* / Q000235:genetics
D002087:Butyrates; D011994:Recombinant Proteins; C033592:4-nitrophenyl butyrate; D008049:Lipase