Molecular mechanism, protein engineering, and application of a thermophilic and halophilic enzyme, thermolysin

考察了热溶素(E)的Tyr、COOH和Lys残基的化学修饰对其活性和亲盐性的影响。酪氨酸和赖氨酸残基的修饰降低了活性，羧基的修饰没有改变活性。经聚乙二醇修饰的E.Lys残基表面电荷减少，其亲盐性降低。E的活性随着修饰程度的增加而增加，当修饰6个赖氨酸残基时，E的活性提高了6倍。PEG修饰使E稳定。聚乙二醇的作用与盐的作用相似。从产E菌的突变体中分离到2个E变异体（T1和T2）。与T1处理一样，E的Val-140转化为Ala，与T2处理一样，Ala-73转化为Val。T1处理的蛋白酶活性是天然E的150%，肽酶活性是35%，T2处理的蛋白酶活性是天然E的10%，但肽酶活性显著提高，达到了1200%。这两个残基对确定E的底物特异性有关键影响。在Asp-73的位置引入一个大的基团，强烈抑制蛋白酶活性，使E转化为肽酶。A73V酶的合成活性是阿斯巴甜前体的10倍。将活性位点锌替换为钴，Co-E的活性是天然E的5倍，表现出亲本E的亲盐性，在4 M NaCl下的活性是天然E的15倍。比较了E与母菌素的活性和结构。它还表现出较强的亲盐性和较高的热稳定性。发现其结构与E的催化结构域相似，并观察到E和基质溶素的亲盐性的分子机制不同；即E的亲盐性是通过稳定底物的过渡态得到的，而基质溶素的亲盐性是通过稳定底物的基态得到的。少

Effect of chemical modification of Tyr, COOH, and Lys residues of thermolysin (E) on its activity and halophilicity was examined. The activity was decreased by the modification of Tyr and Lys residues, while that was not change by that of carboxyls. The decrease in the halophilicity was observed with decreasing the charges on the surface of E.Lys residues were modified by polyethylene glycol (PEG). The activity of E was increased with the degree of the modification, and was 6-times higher when 6 Lys residues were modified. E was stabilized with the PEG modification. The effect of PEG was similar to that of the addition of salts. Two E varinats (T1 and T2) were isolated from mutants of the E-producing bacteria. As with T1, Val-140 of E was converted to Ala, and with T2, Ala-73 was converted to Val. The protease activity of T1 was 150% in comparison with that of the native E, and the peptidase activity was 35% On the other hand, the protease activity of T2 was 10% of that of the native E … More , but the peptidase activity was increased remarkably, to 1,200%. The both residues were shown to have critical influence in determining of the substrate-specificity of E.Introduction of a bulky group into the place of Asp-73 depressed strongly the protease activity, and turned E to a peptidase. The A73V enzyme has 10-times higher activity of synthesis of the aspartame precurser. The active-site zinc was replaced with cobal, and the Co-E was shown to have 5-times higher activity than the native E.Co-E exhibited halophilicity, as the parent E does, and the activity at 4 M NaCl was 15-times higher than that of the native one. The activity-and-structure of E was compared with that of matrilysin. It showed also strong halophilicity, and higher thermal stability. The structure was found to be similar to that of the catalytic domain of E.It was also observed that the molecular mechanism of of halophilicity of E and matrilysin is different ; namely that the halophilicity of E was derived by the stabilizing the transition-state of the substratre, whereas that of matrilysin was by stabilizing its ground-state of the substrate. Less