INACTIVATION OF STAPHYLOCOCCUS-HYICUS LIPASE BY HEXADECYLSULFONYL FLUORIDE - EVIDENCE FOR AN ACTIVE-SITE SERINE

The Staphylococcus hyicus lipase is an acyl hydrolase with broad subst rate specificity including neutral glycerides and phospholipids. To ob tain further insight into the mechanism of action of this enzyme, we t ested several sulfonyl fluorides as active site-directed inhibitors. T he enzyme is resistant to the well-known serine protease/esterase inhi bitor phenylmethanesulfonyl fluoride (PMSF), but is rapidly inactivate d by hexadecylsulfonyl fluoride. The kinetics of inactivation were stu died in Triton X-100 micelles. Inactivation is fast and the rate of in activation is constant over the pH range where this lipase is active. Metal ions like Ca2+ and Sr2+ do not appreciably influence the rate of inactivation, although the enzymatic activity is significantly increa sed, suggesting a structural role for these ions. The S.hyicus lipase contains a consensus sequence G-H/Y-S-X-G. Substitution by site-direct ed mutagenesis of this serine (Ser369) by a cysteine resulted in a mut ant with only 0.2% residual activity. The activity of this mutant coul d not be inhibited with water-soluble sulfhydryl reagents either in th e presence or absence of Triton X-100 micelles. In the presence of Tri ton X-100 micelles, inactivation of the mutant occurred with 4-nitroph enylhexadecyl disulfide (t(1/2) = 125 min) while the wild-type enzyme does not react at all. We conclude that Ser369 is the active site resi due and that in water this residue is inaccessible. Only after interfa cial activation Ser369 (or Cys369) becomes exposed and reacts with irr eversible inhibitors.