Studies on the catalytic mechanism of formate dehydrogenase from Candida boidinii using isotope-labelled substrate and co-enzyme

Abstract

Formate dehydrogenase from Candida boidinii (CbFDH; EC.1.2.1.2) catalyses the formate oxidation and the CO2 reduction using the redox pair of NAD+/NADH as a co-enzyme. Isotope substitution of substrate is a means of addressing the participation of protein motions in biocatalytic reactions. The effects of isotope-labelled NAD+ (NAD+-d4) and deuterium or 13C-labelled-formate (DCOO- or H13COO-) on CbFDH-catalyzed formate oxidation activity were investigated. By using DCOO- as a substrate, the isotope effect was exhibited in the process of CbFDH-catalyzed formate oxidation and the reaction rate was reduced by 38 % compared to the system using non-isotope-labelled formate. Moreover, the isotope effect also was strongly exhibited using both of NAD+-d4 and DCOO- in the formate oxidation with CbFDH and the reaction rate was reduced by 28 % compared to the system using non-isotope-labelled NAD+ and formate. On the other hand, it was clarified that there is no significantly affect the formate oxidation reaction catalyzed by CbFDH using 13C-labeled formate as a substrate. From these results, it was suggested that C-H bond cleavage of formate and nucleophilic attack of hydrogen on NAD+ dominate the reaction rate in CbFDH-catalyzed formate oxidation. In addition, the hydrogen bonds formed between formate, NAD+ and water molecules in the substrate binding site of CbFDH was also important in the formate oxidation to CO2.