Simple alkanethiol groups for temporary blocking of sulfhydryl groups of enzymes.
Simple alkanethiol groups for temporary blocking of sulfhydryl groups of enzymes.
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简单的烷硫醇基团用于暂时封闭酶的巯基。
DOI：
10.1021/bi00685a601
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发表时间：
1975
期刊：
影响因子：
2.9
通讯作者：
G. L. Kenyon
中科院分区：
文献类型：
--
作者：
D. J. Smith;E. Maggio;G. L. Kenyon
用于暂时阻断酶的活性或可接近的巯基的新试剂已经开发出来。这些试剂，要么是烷基烷硫醇磺酸盐，要么是烷氧羰基烷基二硫化物，快速定量地将各种RS-基团置于巯基上，生成混合二硫化物。在所有情况下，原生酶都可以用二硫苏糖醇或-巯基乙醇再生。一般来说，临时阻断基团也对通常具有抑制作用的硫醇阻断剂提供全面保护。当RS-组与兔肌肌酸激酶（EC 2.7.3.2）结合时，随着体积的增大，残余活性呈降低趋势。用14CH3HgC1处理天然肌酸激酶，导致每个亚基有超过1等量的CH3Hg-基团掺入。该CH3Hg阻断酶具有完全活性，阻断组对碘乙酰胺无保护作用。这些结果表明CH3Hg-和RS-基团修饰了酶上的两个不同的巯基。用过量的甲基乙基磺酸盐处理木瓜蛋白酶（EC 3.4.4.10）。实验结果表明，每mol活性酶可掺入1等量的CH3S-。临时阻断基团对通常具有抑制作用的5,5′-二硫代比斯（2-硝基苯甲酸）具有完全的保护作用。用甲乙磺酸甲酯滴定兔肌甘油醛-3-磷酸脱氢酶（EC 1.2.1.12）时，发现每个亚基有两个巯基被修饰，其中一个比另一个快得多。如果有人推断滴定曲线的初始斜率，酶的失活将在修饰每个亚基的单个半胱氨酸残基后完成。
New reagents for the temporary blocking of active or accessible sulfhydryl groups of enzymes have been developed. These reagents, which are either alkyl alkanethiolsulfonates or alkoxycarbonylalkyl disulfides, rapidly and quantitatively place various RS- groups on the sulfhydryls to generate mixed disulfides. In all cases native enzymes can be regenerated with either dithiothreitol or beta-mercaptoethanol. In general the temporary blocking groups also afford total protection against normally inhibitory thiol blocking agents. When RS- groups were attached to rabbit muscle creatine kinase (EC 2.7.3.2), a trend toward lower residual activities with increasing bulk was observed. Treatment of the native creatine kinase with 14CH3HgC1 led to incorporation of greater than 1 equiv of CH3Hg- group per subunit. This CH3Hg- blocked enzyme was fully active, and the blocking group afforded no protection against iodoacetamide. These results suggest that CH3Hg- and the RS- groups are modifying two different sulhydryl groups on the enzyme. When papain (EC 3.4.4.10) was treated with excess methyl methanethiolsulfonate. complete and rapid inhibition was observed, and 1 equiv of CH3S- was incorporated/mol of active enzyme. Complete protection against normally inhibitory 5,5'-dithiobis(2-nitrobenzoic acid) was afforded by the temporary blocking group. When rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) was titrated with methyl methanethiolsulfonate, two sulfhydryl groups per subunit were found to be modified, one much more rapidly than the other. If one extrapolates the initial slope of the titration curve, the inactivation of the enzyme would be complete after modification of a single cysteinyl residue per subunit.