Inouye K, Lee S B, Tonomura B
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Kyoto, 606-8502, Japan.
J Biochem. 1998 Jul;124(1):72-8. doi: 10.1093/oxfordjournals.jbchem.a022099.
Thermolysin is remarkably activated in the presence of high concentrations (1-5 M) of neutral salts and its activity is enhanced 15 times by 4 M NaCl at pH 7.0 and 25 degrees C [Inouye, K. (1992) J. Biochem. 112, 335-340]. In this study, the effects of nitration and amination of tyrosyl residues in thermolysin on its halophilic properties were examined. Nitration and successive amination inactivate thermolysin progressively as the degree of modification increases. When 16 tyrosyl residues were nitrated, the activity decreased to 10% of that of the native enzyme, whereas it recovered to 30% when they were aminated. The decrease in the activity by the nitration and amination was shown to be brought about only by a decrease in the molecular activity, kcat; the Michaelis constant, Km, was unaltered. When 14 tyrosyl residues of thermolysin were nitrated, the degree of activation by 4 M NaCl at pH 7.0 decreased from 15 to 10, and this decreased further to 5 when the pH of the reaction medium was raised to 8.5. However, when the nitrated tyrosyl residues were reduced to aminotyrosyl residues, the degree of activation was restored to that of the native enzyme. The change in the degree of activation by nitration and amination of thermolysin could be due to the change in the ionization of tyrosyl residues, and it was suggested that removing negative charges from tyrosyl residues of thermolysin enhances its halophilicity.
嗜热菌蛋白酶在高浓度（1 - 5 M）中性盐存在时会显著活化，在pH 7.0和25℃条件下，4 M NaCl可使其活性增强15倍[猪谷，K.（1992年）《生物化学杂志》112卷，335 - 340页]。在本研究中，检测了嗜热菌蛋白酶中酪氨酸残基的硝化和胺化对其嗜盐特性的影响。随着修饰程度增加，硝化和连续胺化会逐渐使嗜热菌蛋白酶失活。当16个酪氨酸残基被硝化时，活性降至天然酶的10%，而胺化后则恢复至30%。硝化和胺化导致的活性降低仅表现为分子活性kcat的下降；米氏常数Km未改变。当嗜热菌蛋白酶的14个酪氨酸残基被硝化时，在pH 7.0条件下4 M NaCl的活化程度从15降至10，当反应介质pH升至8.5时，活化程度进一步降至5。然而，当硝化的酪氨酸残基还原为氨基酪氨酸残基时，活化程度恢复至天然酶水平。嗜热菌蛋白酶硝化和胺化后活化程度的变化可能是由于酪氨酸残基电离的改变，并且有人提出从嗜热菌蛋白酶的酪氨酸残基上去除负电荷会增强其嗜盐性。
