Triple Isotope Effects Support Concerted Hydride and Proton Transfer and Promoting Vibrations in Human Heart Lactate Dehydrogenase.
Triple Isotope Effects Support Concerted Hydride and Proton Transfer and Promoting Vibrations in Human Heart Lactate Dehydrogenase.
复制标题
三重同位素效应支持协同氢化物和质子转移并促进人心脏乳酸脱氢酶的振动。
DOI：
10.1021/jacs.6b09049
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发表时间：
2016-11-16
影响因子：
15
通讯作者：
Schramm VL
中科院分区：
文献类型：
--
作者：
Wang Z;Chang EP;Schramm VL
过渡路径采样模拟提出，人心脏乳酸脱氢酶 (LDH) 在飞秒 (fs) 到皮秒 (ps) 时间尺度上利用蛋白质促进振动 (PPV) 来促进跨越化学屏障。这种化学屏障涉及氢化物和质子转移到丙酮酸以形成 L-乳酸，使用还原型烟酰胺腺嘌呤二核苷酸 (NADH) 作为辅因子。在这里，我们报告了来自三种类型同位素效应实验的实验证据，这些实验证据支持促进振动与势垒穿越的耦合以及氢化物和质子转移的一致性。我们制备了天然（轻）LDH 和用 13C、15N 和不可交换 2H (D) 标记的重 LDH，以干扰预测的 PPV。重LDH在单周转实验中减缓了化学反应，支持PPV对过渡态形成的贡献。在结合 NADD 和 D2O 的双标记实验中，[4-2H]NADH (NADD) 动力学同位素效应和 D2O 溶剂同位素效应均有所增加，轻 LDH 和重 LDH 均保持这种模式。这些同位素效应支持轻 LDH 和重 LDH 的协同氢化物和质子转移。尽管重LDH中过渡态跨越势垒的概率降低，但氢化物-质子转移反应的协同机制并未改变。本研究利用三重同位素效应来解析 LDH 的化学机制，并建立 fs-ps 蛋白质动力学与屏障跨越的耦合。
Transition path sampling simulations have proposed that human heart lactate dehydrogenase (LDH) employs protein promoting vibrations (PPVs) on the femtosecond (fs) to picosecond (ps) time scale to promote crossing of the chemical barrier. This chemical barrier involves both hydride and proton transfers to pyruvate to form l-lactate, using reduced nicotinamide adenine dinucleotide (NADH) as the cofactor. Here we report experimental evidence from three types of isotope effect experiments that support coupling of the promoting vibrations to barrier crossing and the coincidence of hydride and proton transfer. We prepared the native (light) LDH and a heavy LDH labeled with 13C, 15N, and nonexchangeable 2H (D) to perturb the predicted PPVs. Heavy LDH has slowed chemistry in single turnover experiments, supporting a contribution of PPVs to transition state formation. Both the [4-2H]NADH (NADD) kinetic isotope effect and the D2O solvent isotope effect were increased in dual-label experiments combining both NADD and D2O, a pattern maintained with both light and heavy LDHs. These isotope effects support concerted hydride and proton transfer for both light and heavy LDHs. Although the transition state barrier-crossing probability is reduced in heavy LDH, the concerted mechanism of the hydride–proton transfer reaction is not altered. This study takes advantage of triple isotope effects to resolve the chemical mechanism of LDH and establish the coupling of fs-ps protein dynamics to barrier crossing.