Abstract
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 pK(a) shift induced by the electrostatics of a neighboring histidine residue (H110), whose protonation state change modulates the pK(a) 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.