STUDYING THE ACTIVE-SITE POCKET OF STAPHYLOCOCCUS-HYICUS LIPASE BY SITE-DIRECTED MUTAGENESIS

Site-directed mutagenesis of a previously constructed, recombinant Sta phlococcus hyicus lipase (49 kDa) showed that Va1363 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), respect ively. In comparison with wild type enzyme, a G371A enzyme showed a 40 % decrease in the k(m), suggesting that G371 was important for substra te-binding specificity. Site-directed mutagenesis of the active site A sp559 revealed that in comparison with wild type enzyme, a D559E enzym e 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 compone nt of the catalytic triad, was involved in substrate-specificity. (C) 1996 Academic Press, Inc.