Leuveling Tjeenk M, Bulsink Y B, Slotboom A J, Verheij H M, de Haas G H, Demleitner G, Götz F
Department of Enzymology and Protein Engineering, University of Utrecht, The Netherlands.
Protein Eng. 1994 Apr;7(4):579-83. doi: 10.1093/protein/7.4.579.
The Staphylococcus hyicus lipase is an acyl hydrolase with broad substrate specificity including neutral glycerides and phospholipids. To obtain further insight into the mechanism of action of this enzyme, we tested several sulfonyl fluorides as active site-directed inhibitors. The enzyme is resistant to the well-known serine protease/esterase inhibitor phenylmethanesulfonyl fluoride (PMSF), but is rapidly inactivated by hexadecylsulfonyl fluoride. The kinetics of inactivation were studied in Triton X-100 micelles. Inactivation is fast and the rate of inactivation 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 increased, suggesting a structural role for these ions. The S. hyicus lipase contains a consensus sequence G-H/Y-S-X-G. Substitution by site-directed mutagenesis of this serine (Ser369) by a cysteine resulted in a mutant with only 0.2% residual activity. The activity of this mutant could not be inhibited with water-soluble sulfhydryl reagents either in the presence or absence of Triton X-100 micelles. In the presence of Triton X-100 micelles, inactivation of the mutant occurred with 4-nitrophenylhexadecyl disulfide (t1/2 = 125 min) while the wild-type enzyme does not react at all. We conclude that Ser369 is the active site residue and that in water this residue is inaccessible. Only after interfacial activation Ser369 (or Cys369) becomes exposed and reacts with irreversible inhibitors.
猪葡萄球菌脂肪酶是一种酰基水解酶，具有广泛的底物特异性，包括中性甘油酯和磷脂。为了进一步深入了解这种酶的作用机制，我们测试了几种磺酰氟作为活性位点导向抑制剂。该酶对著名的丝氨酸蛋白酶/酯酶抑制剂苯甲基磺酰氟（PMSF）具有抗性，但能被十六烷基磺酰氟迅速灭活。在 Triton X - 100 胶束中研究了灭活动力学。灭活速度很快，且在该脂肪酶具有活性的 pH 范围内，灭活速率是恒定的。像 Ca2+ 和 Sr2+ 这样的金属离子虽然能显著提高酶活性，但对灭活速率没有明显影响，这表明这些离子具有结构作用。猪葡萄球菌脂肪酶含有一个共有序列 G - H/Y - S - X - G。通过定点诱变将该丝氨酸（Ser369）替换为半胱氨酸，得到一个残余活性仅为 0.2% 的突变体。无论有无 Triton X - 100 胶束，该突变体的活性都不能被水溶性巯基试剂抑制。在 Triton X - 100 胶束存在的情况下，4 - 硝基苯基十六烷基二硫化物能使该突变体失活（t1/2 = 125 分钟），而野生型酶根本不发生反应。我们得出结论，Ser369 是活性位点残基，在水中该残基无法接近。只有在界面激活后，Ser369（或 Cys369）才会暴露并与不可逆抑制剂发生反应。
