Formate Dehydrogenase: The Role of the AMP Cofactor Fragment in Stabilization of the Transition State for Enzyme-Catalyzed Hydride Transfer

We report experiments that probe the role of the NAD+-driven protein conformational change in the formate dehydrogenase (FDH)-catalyzed hydride transfer. The binding interactions between FDH and the ADP fragment of NAD+ provide an 11.9 kcal/mol stabilization of the transition state for FDH-catalyzed hydride transfer from formate to NAD+ and a 7.9 kcal/mol stabilization of the complex between FDH and the putative transition state analogue azide anion. The binding interactions between FDH and the AMP cofactor piece likewise provide a 5.6 kcal/mol stabilization of the transition state for FDH-catalyzed hydride transfer from formate to nicotinamide riboside (NR) and a 1.4 kcal/mol stabilization of the complex between FDH and the azide anion. The results provide support for the conclusion that binding of NAD+ or the AMP cofactor fragment to FDH drives a change in protein conformation from a flexible open conformation to the tight closed conformation that locks the active site side chains into positions that provide optimal stabilization of both the hydride transfer transition state and the azide anion mimic for this transition state.