On the reactivity of the thiol group of thiolsubtilisin.
On the reactivity of the thiol group of thiolsubtilisin.
复制标题
关于硫醇枯草杆菌蛋白酶的硫醇基团的反应性。
DOI：
--
复制
发表时间：
1973
期刊：
影响因子：
--
通讯作者：
E. Moravcsik
中科院分区：
文献类型：
--
作者：
L. Polgár;P. Halász;E. Moravcsik
1
硫代枯草杆菌素与碘乙酰胺的反应表明，在碱性pH范围内，该酶的-SH基团类似于简单的硫醇化合物，如巯基乙酸酯。另一方面，与巯基乙酸酯相比，在中性pH附近，该酶表现出更强的反应活性。根据在2H2O中的测量，得出结论：在硫代枯草杆菌中形成了硫醇-咪唑离子对，这解释了在中性pH附近反应活性增强的原因。
2.
与硫代乙酸乙酯相比，碘乙酰胺与酶的-SH基团的反应速率相对高于溴、氯和氟乙酰胺。这可能是由于在二氧六环存在下，巯基乙酸酯与碘和氯乙酰胺的烷基化反应表明，酶表面的-SH基团周围的环境不如水中的极性环境所致。
3.
通过硫代枯草杆菌蛋白与对映体的烷基化反应，可以在pH·9以上建立常规物理方法无法测量的活性部位的轻微构象变化，而整个蛋白质结构在pH·11以下是稳定的。D-2-溴-正丁酰胺与硫代枯草杆菌蛋白的反应速度是D-2-溴丙酰胺的60倍。这表明酶与底物的甲基之间的疏水相互作用可以使反应速度提高一个数量级以上。活性中心的模型构建表明，如果烷基化试剂的位置合适，硫代枯草杆菌的Ala-152和Thr-220可能与D-2-溴-正丁酰胺形成van der Waals相互作用。D-2-溴丙酰胺和硫代枯草杆菌之间不可能发生这种相互作用。
1
The reaction of thiolsubtilisin with iodoacetamide shows that in the alkaline pH-range the –SH group of the enzyme reacts like a simple thiol compound such as mercaptoacetate. On the other hand, around neutral pH the enzyme displays enhanced reactivity compared with that of mercaptoacetate. On the basis of measurements in 2H2O it is concluded that a mercaptide-imidazolium ion-pair is formed in thiolsubtilisin and this accounts for the enhanced reactivity around neutral pH.
2
Iodoacetamide reacts with the –SH group of the enzyme, as compared to mercaptoacetate, at a relatively higher rate than bromo-, chloro-, and fluoroacetamide. This might be due to the less polar environment around the –SH group on the surface of the enzyme than in water, as indicated by alkylation of mercaptoacetate with iodo- and chloroacetamide in the presence of dioxane.
3
By alkylating thiolsubtilisin with enantiomeric reactants, a slight conformational change at the active site, not measurable with ordinary physical methods, could be established above pH · 9, whereas the overall protein structure is stable up to pH · 11. D-2-Bromo-n-butyramide reacts with thiolsubtilisin about 60 times as fast as D-2-bromopropionamide. This indicates that hydrophobic interaction between the enzyme and a methyl group of the substrate can enhance the reaction rate by more than one order of magnitude. Model building of the active site suggests that Ala-152 and Thr-220 of thiolsubtilisin may form van der Waals interactions with D-2-bromo-n-butyramide if the alkylating agent is properly positioned for reaction. No such interaction is possible between D-2-bromopropionamide and thiolsubtilisin.