Jakubowski H
Department of Microbiology & Molecular Genetics, UMDNJ--New Jersey Medical School, Newark 07103, USA
Biochemistry. 1996 Jun 25;35(25):8252-9. doi: 10.1021/bi960344v.
The active site of methionyl-tRNA synthetase (MetRS) possesses two functions: synthetic, which provides Met-tRNA for protein synthesis, and editing, which rejects inadvertently misactivated homocysteine. During editing, the side chain -SH group of homocysteine reacts with its activated carboxyl group forming a cyclic thioester, homocysteine thiolactone. As shown here, the side chain -SH and the activated carboxyl groups do not need to be present on the same molecule for the editing to occur. Thioester formation occurs when a thiol and activated methionine, in the form of Met-tRNA, are incubated with MetRS. Depending on the structure of thiols, methionine thioesters may undergo secondary acyl transfer reactions to cis amino, hydroxy, or carboxyl groups which yield methionine dipeptides, esters, or anhydrides, respectively. At saturating thiol concentrations, formation of some thiol derivatives of methionine is as fast as formation of homocysteine thiolactone. Thiol specificity of the reaction and noncompetitive inhibition by the cognate methionine, as well as structure-function studies of active site MetRS mutants, all indicate that there is a specific -SH binding subsite, distinct from the methionine binding subsite, in the synthetic/editing active site of MetRS.
甲硫氨酰 - tRNA合成酶（MetRS）的活性位点具有两种功能：合成功能，为蛋白质合成提供甲硫氨酰 - tRNA；编辑功能，剔除意外被错误激活的同型半胱氨酸。在编辑过程中，同型半胱氨酸的侧链 -SH基团与其被激活的羧基反应，形成环状硫酯，即同型半胱氨酸硫内酯。如本文所示，编辑过程发生时，侧链 -SH和被激活的羧基并不需要存在于同一分子上。当硫醇与以甲硫氨酰 - tRNA形式存在的被激活的甲硫氨酸一起与MetRS温育时，会发生硫酯形成反应。根据硫醇的结构，甲硫氨酸硫酯可能会发生二级酰基转移反应，分别生成甲硫氨酸二肽、酯或酸酐的顺式氨基、羟基或羧基。在硫醇浓度饱和时，甲硫氨酸的一些硫醇衍生物的形成速度与同型半胱氨酸硫内酯的形成速度一样快。该反应的硫醇特异性以及同源甲硫氨酸的非竞争性抑制作用，以及活性位点MetRS突变体的结构 - 功能研究，均表明在MetRS的合成/编辑活性位点中存在一个与甲硫氨酸结合亚位点不同的特定 -SH结合亚位点。
