Utilization of Cofactor Binding Energy for Enzyme Catalysis: Formate Dehydrogenase-Catalyzed Reactions of the Whole NAD Cofactor and Cofactor Pieces

Abstract

The pressure to optimize enzymatic rate accelerations has driven the evolution of the induced-fit mechanism for enzyme catalysts where the binding interactions of nonreacting phosphodianion or adenosyl substrate pieces drive enzyme conformational changes to form protein substrate cages that are activated for catalysis. We report the results of experiments to test the hypothesis that utilization of the binding energy of the adenosine 5′-diphosphate ribose (ADP-ribose) fragment of the NAD cofactor to drive a protein conformational change activates Candida boidinii formate dehydrogenase (CbFDH) for catalysis of hydride transfer from formate to NAD+. The ADP-ribose fragment provides a >14 kcal/mol stabilization of the transition state for CbFDH-catalyzed hydride transfer from formate to NAD+. This is larger than the ca. 6 kcal/mol stabilization of the ground-state Michaelis complex between CbFDH and NAD+ (K NAD = 0.032 mM). The ADP, AMP, and ribose 5′-phosphate fragments of NAD+ activate CbFDH for catalysis of hydride transfer from formate to nicotinamide riboside (NR). At a 1.0 M standard state, these activators stabilize the hydride transfer transition states by ≈5.5 (ADP), 5.5 (AMP), and 4.4 (ribose 5′-phosphate) kcal/mol. We propose that activation by these cofactor fragments is partly or entirely due to the ion-pair interaction between the guanidino side chain cation of R174 and the activator phosphate anion. This substitutes for the interaction between the α-adenosyl pyrophosphate anion of the whole NAD+ cofactor that holds CbFDH in the catalytically active closed conformation.

Glycerol 3-Phosphate Dehydrogenase: Role of the Protein Conformational Change in Activation of a Readily Reversible Enzyme-Catalyzed Hydride Transfer Reaction

“…Figure was developed to rationalize activation of reactions of truncated substrates catalyzed by GPDH, triosephosphate isomerase (TIM), , orotidine 5′-phosphate decarboxylase (OMPDC) by phosphite, and other dianions. It has been generalized to other phosphite-dianion-activated enzyme-catalyzed reactions, , and to enzymatic reactions activated by other substrate fragments. , The critical feature is the utilization of dianion binding energy to stabilize the active closed form of the E · cofactor complex ( E C ). This is consistent with a large body of experimental results from studies on GPDH. − However, the mechanism from Schemes and has not been compared critically with a second proposed mechanism for dianion activation (Scheme ).…”

“…Such strong fragment activation is a property of Nature’s most proficient enzyme catalysts. For example, the adenosine fragment of AMP activates adenylate kinase for catalysis of phosphoryl transfer from ATP to phosphite and other dianions, , and the ADP fragment of NAD activates formate dehydrogenase for catalysis of hydride transfer from formate to nicotinamide riboside …”