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

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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.