Alkylation and identification of the histidine residues at the active site of ribonuclease.
Alkylation and identification of the histidine residues at the active site of ribonuclease.
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核糖核酸酶活性位点组氨酸残基的烷基化和鉴定。
DOI：
10.1016/s0021-9258(19)67986-x
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发表时间：
1963
期刊：
影响因子：
--
通讯作者：
S. Moore
中科院分区：
文献类型：
--
作者：
A. M. Crestfield;W. Stein;S. Moore
碘乙酸酯和溴乙酸酯与核糖核酸酶的反应已经为该酶的化学结构与催化活性之间的关系提供了一些信息。本文和下一篇论文的目的是扩展这一信息。在早期的研究中，Gundlach、Stein和Moore(1)表明，尽管赖氨酸、甲硫氨酸或组氨酸残基上可能发生烷基化，但碘乙酸酯最特异的抑制作用主要涉及在pH 5.5至pH 6的条件下形成含有I(或3)羧甲基组氨酸的单一残基的衍生物。Rarnard和R.11.Stein(2，3)独立地研究了一种类似类型的反应，在该反应中，C%卤代溴代乙酸酯是烷化剂。根据放射性测量，他们得出结论，每个酶分子中都引入了一个羧基。Rarnard和Stein对反应混合物中的所有蛋白质进行氧化、胰凝乳凝乳酶的水解和纸电泳法分离产物后，发现了一个单一的放射性多肽；根据多肽的氨基酸组成，他们得出结论：在核糖核酸酶中烷基化的组氨酸是119位。Gundlach等人。还观察到在pH 5.5时形成了少量的第二个非活性衍生物，其在Amberlite IRC-50上的移动速度较慢。目前的来文与CHA有关。当核糖核酸酶中给定咪唑环被碘乙酸酯烷基化时，反应的两个产物的异构化，确定在每个情况下改变的氨基酸残基的位置，以及确定两个结构异构体(I-羧甲基或3-羧甲基组氨酸)中的哪一个形成。
The reactions of iodoacetate and bromoacetate with ribonuclease have already provided some information on the relationship between the chemical structure and the catalytic a (*-tivity of the enzyme. It is the purpose of this and the following paper to extend this information. In earlier studies, Gundlach, Stein, and Moore (1) showed that although alkylation at lysine, mrthioninc, or histidine residues could occur, the most specific inartivation by iodoacetate involved predominantly the formation at pH 5.5 to pH 6 of a derivative which contained a single residue of I (or 3) carboxymethylhistidinc. Independently, Rarnard and R. 11. Stein (2, 3) studied a similar type of reaction in which C% brled bromoacctate was the alkylating agent. From radioactivity measurements they concluded that one carboxymcthyl group per molecule of enzyme had been introduced. Upon oxidation of all of the protein in the reaction mixture, hydrolysis\yith chymotrypsin, and separation of the productas by paper electrophoresis, Rarnard and Stein found a single radioactive peptidc; from the amino acid composition of the peptide they concluded that the histidine which had been alkylated in ribonuclease was the one at position 119. Gundlach et al. also observed the formation at pH 5.5 of a small amount of a second inactive derivative which had a slower rate of travel on Amberlite IRC-50. The present communication is concerned with the cha. racterization of the two products of the reaction, the establishment of the position of the amino acid residues altered in each instance, and the determination of which of the two structural isomers (I-carboxymethyl- or 3-carboxymethylhistidine) is formed when a given imidazole ring in ribonuclease is alkylated by iodoacetate.