Uranga Jon, Rabe von Pappenheim Fabian, Tittmann Kai, Mata Ricardo A
Institute of Physical Chemistry, Georg-August Universität Göttingen, Tammannstraße 6, 37077 Göttingen, Germany.
Department of Molecular Enzymology, Göttingen Center of Molecular Biosciences, Georg-August Universität Göttingen, Julia-Lermonotowa-Weg 3, D-37077 Göttingen, Germany.
J Phys Chem B. 2023 Sep 25;127(44):9423-32. doi: 10.1021/acs.jpcb.3c03137.
The activation mechanism of thiamine diphosphate (ThDP) in enzymes has long been the subject of intense research and controversial discussion. Particularly contentious is the formation of a carbene intermediate, the first one observed in an enzyme. For the formation of the carbene to take place, both intramolecular and intermolecular proton transfer pathways have been proposed. However, the physiologically relevant pH of ThDP-dependent enzymes around neutrality does not seem to be suitable for the formation of such reactive chemical species. Herein, we investigate the general mechanism of activation of the ThDP cofactor in human transketolase (TKT), by means of electronic structure methods. We show that in the case of the human TKT, the carbene species is accessible through a p shift induced by the electrostatics of a neighboring histidine residue (H110), whose protonation state change modulates the p of ThDP and suppresses the latter by more than 6 pH units. Our findings highlight that ThDP enzymes activate the cofactor beyond simple geometric constraints and the canonical glutamate. Such observations in nature can pave the way for the design of biomimetic carbene catalysts and the engineering of tailored enzymatic carbenes.
硫胺素二磷酸（ThDP）在酶中的激活机制长期以来一直是深入研究和有争议讨论的主题。特别有争议的是卡宾中间体的形成，这是在酶中观察到的第一个此类中间体。为了发生卡宾的形成，已经提出了分子内和分子间质子转移途径。然而，ThDP依赖性酶在中性附近的生理相关pH似乎不适合形成这种活性化学物质。在此，我们通过电子结构方法研究了人转酮醇酶（TKT）中ThDP辅因子的一般激活机制。我们表明，在人TKT的情况下，卡宾物种可通过相邻组氨酸残基（H110）的静电作用诱导的p位移获得，其质子化状态变化调节ThDP的p并将其抑制超过6个pH单位。我们的研究结果突出表明，ThDP酶对辅因子的激活超出了简单的几何限制和典型的谷氨酸。自然界中的此类观察结果可为仿生卡宾催化剂的设计和定制酶促卡宾的工程化铺平道路。
