Structure and reactivity of intermediates in the carboligation of ThDP-dependent enzymes

除了在细胞代谢中的功能外，依赖于硫胺素的二磷酸(ThDP)酶最近已经成为2-酮酸、醛和糖的对映和立体选择性碳配基反应的有力工具，以产生各种药学上有意义的化合物。对于破译碳化反应和底物专一性的一个关键问题涉及分子确定剂的分析，该分子确定剂定义了供体和受体底物的结合位置及其专一性，以及来自供体的中心碳负离子/烯胺中间体的动力学(去)稳定性。以典型的ThDP依赖酶丙酮酸脱羧酶(PDC)和转酮醇酶(TK)为例，我们的研究旨在从分子水平上推断途径外(PDC)或途径上(TK)碳化的机理。根据现有的动力学、光谱和结构信息，应通过微观动力学和热力学分析严格检查野生型酶和具有假定改变的底物结合袋和动力学稳定的碳负离子/烯胺中间体的变体中的碳化作用。在适当的时候，碳化的关键中间体将通过X射线结晶学进行结构表征。总之，应该开发一个机械框架，允许合理地设计具有明确反应和底物特异性的量身定制的ThDP依赖酶。

Besides their well-characterized functions in cellular metabolism, thiamine diphosphate (ThDP)-dependent enzymes have recently emerged as powerful tools for the enantio- and stereoselective carboligation of 2-ketoacids, aldehydes and sugars to yield a variety of pharmaceutically interesting compounds. A crucial issue for deciphenng the reaction and substrate specificity of carboligation relates to the analysis of molecular detenninants that define the binding sites and therewith specificity of donor and acceptor substrates, and the kinetic (de)-stabilisation of the central carbanion/enamine intermediate deriving from the donor. Exemplified for the prototypical ThDP-dependent enzymes pyruvate decarboxylase (PDC) and transketolase (TK), our studies are aimed to - on a molecular level - deduce mechanistic principles by which off-pathway (PDC) or on-pathway (TK) carboiigation are conferred. On the basis of available kinetic, spectroscopic and structural information, carboligation in wild-type enzymes and variants with putatively altered substrate binding pockets and a kinetically stabilized carbanion/enamine intermediate shall be rigorously examined by means of microscopic kinetic and thermodynamic analysis. When appropriate, key intermediates of carboligation will be structurally characterized by X-ray crystallography. In conclusion, a mechanistic framework shall be developed that allows to rationally design tailor-made ThDP-dependent enzymes with defined reaction and substrate specificity.