Evidence for dramatic acceleration of a C-H bond ionization rate in thiamin diphosphate enzymes by the protein environment.
Evidence for dramatic acceleration of a C-H bond ionization rate in thiamin diphosphate enzymes by the protein environment.
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蛋白质环境显着加速硫胺素二磷酸酶中 C-H 键电离速率的证据。
DOI：
10.1021/bi047696j
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Jordan,Frank
中科院分区：
文献类型：
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作者：
Zhang,Sheng;Zhou,Leon;Nemeria,Natalia;Yan,Yan;Zhang,Zhen;Zou,Yu;Jordan,Frank
通过使用中间体C2α-羟乙基硫胺素二磷酸（HEThDP）与大肠杆菌丙酮酸脱氢酶复合物及其E1亚基（PDHc-E1），实验验证了以下假设：硫胺素二磷酸依赖性酶通过提供有助于稳定不稳定的两性离子/偶极中间体（包括所有此类酶上存在的烯胺/C2α-碳负离子）的蛋白质环境来实现其催化速率加速的显著部分。使用预稳态和稳态方法，表明HEThDP是C2α-H键电离后该酶的底物。然后进行实验以测量从HEThDP-d4的C2α位进行D → H交换的PDHc-E1催化的预稳态速率常数，作为烯胺形成的指示。重要的是，该酶将该键的电离速率加速107倍，对应于酶对烯胺中间体的10 kcal/mol稳定化。这一发现可能是硫胺素二磷酸酶的一般特征。
The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment that helps to stabilize unstable zwitterionic/dipolar intermediates (including the enamine/C2α-carbanion present on all such enzymes) was tested experimentally using the intermediate C2α-hydroxyethylthiamin diphosphate (HEThDP) with theEscherichia colipyruvate dehydrogenase complex and its E1 subunit (PDHc-E1). Using pre-steady-state and steady-state methods, it was shown that HEThDP is a substrate for this enzyme after ionization of the C2α-H bond. An experiment was then carried out to measure the PDHc-E1 catalyzed pre-steady-state rate constant for the D → H exchange from the C2α position of HEThDP-d4, as an indicator of the formation of the enamine. Importantly, the enzyme accelerates the rate of ionization of this bond by a factor of 107, corresponding to a 10 kcal/mol stabilization of the enamine intermediate by the enzyme. This finding is likely a general feature of thiamin diphosphate enzymes.