Hydrogen tunneling in adenosylcobalamin-dependent glutamate mutase: evidence from intrinsic kinetic isotope effects measured by intramolecular competition.
Hydrogen tunneling in adenosylcobalamin-dependent glutamate mutase: evidence from intrinsic kinetic isotope effects measured by intramolecular competition.
复制标题
腺苷钴胺依赖性谷氨酸变位酶中的氢隧道：通过分子内竞争测量的内在动力学同位素效应的证据。
DOI：
10.1021/bi1001695
复制
发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Marsh,ENeilG
中科院分区：
文献类型：
--
作者：
Yoon,Miri;Song,Hangtian;Håkansson,Kristina;Marsh,ENeilG
底物和辅酶之间的氢原子转移反应是所有腺苷钴胺素依赖性酶的关键机制特征。对于这些酶之一，谷氨酸酯酶，我们已经研究了氢隧穿是否作出了重大贡献的机制，通过检查与转移的氢原子从甲基天冬氨酸辅酶的氘动力学同位素效应的温度依赖性。为此，我们设计了一种新的分子内竞争实验，使我们能够测量固有的动力学同位素效应，即使氢转移可能不是速率决定的。从动力学同位素效应的Arrhenius图来看，指前因子的比值（AH/AD）为0.17 ± 0.04，同位素效应对活化能[ΔEa（D−H）]的影响为1.94 ± 0.13 kcal/mol。结果表明，一个显着程度的氢隧道发生在谷氨酸盐，即使内在的动力学同位素效应是很好的半经典极限内，远小于那些测得的其他cDNACbl酶和模型反应，其中氢隧道已牵连。
Hydrogen atom transfer reactions between the substrate and coenzyme are key mechanistic features of all adenosylcobalamin-dependent enzymes. For one of these enzymes, glutamate mutase, we have investigated whether hydrogen tunneling makes a significant contribution to the mechanism by examining the temperature dependence of the deuterium kinetic isotope effect associated with the transfer of a hydrogen atom from methylaspartate to the coenzyme. To do this, we designed a novel intramolecular competition experiment that allowed us to measure the intrinsic kinetic isotope effect, even though hydrogen transfer may not be rate-determining. From the Arrhenius plot of the kinetic isotope effect, the ratio of the pre-exponential factors (AH/AD) was 0.17 ± 0.04 and the isotope effect on the activation energy [ΔEa(D−H)] was 1.94 ± 0.13 kcal/mol. The results imply that a significant degree of hydrogen tunneling occurs in glutamate mutase, even though the intrinsic kinetic isotope effects are well within the semiclassical limit and are much smaller than those measured for other AdoCbl enzymes and model reactions for which hydrogen tunneling has been implicated.