Enzyme Architecture: Activation of Phosphite Dehydrogenase-Catalyzed Hydride Transfer by NAD⁺ Cofactor Fragments

We report the results of experiments to test the hypothesis that binding energy from the adenosine diphosphate (ADP) fragment of the NAD⁺ cofactor is utilized to drive a protein conformational change that activates phosphite dehydrogenase (PTDH) for catalysis of hydride transfer from phosphite to NAD⁺. The ADP fragment of the NAD⁺ cofactor provides >8.5 kcal/mol stabilization of the transition state for PTDH-catalyzed hydride transfer. The ADP and AMP fragments of NAD⁺ activate PTDH for catalysis of hydride transfer from phosphite to nicotinamide riboside (NR). At a 1.0 M standard state these activators stabilize the hydride transfer transition state by 5.1 (ADP) and 2.7 (AMP) kcal/mol, so the activation is due to protein interactions with both the α- and β-ADP phosphates. There is no detectable stabilization of the transition state for PTDH-catalyzed hydride transfer to NR by the adenosine fragment of NAD⁺. Activation is proposed to result from stabilization of the closed form of PTDH by a cation–anion pair with the K76 side chain that bridges the α- and β-phosphates of NAD⁺. By comparison, the activation of formate dehydrogenase- and glycerol phosphate dehydrogenase-catalyzed hydride transfer by ADP is from enzyme interactions with the α-phosphate of ADP, with little or no contribution from the β-phosphate. These results show a diversity in the evolution of enzyme-activating conformational changes for dehydrogenase-catalyzed hydride transfer reactions.