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

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+ (KNAD = 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.

Phosphodianion Activation of Enzymes for Catalysis of Central Metabolic Reactions.

The activation barriers ΔG⧧ for kcat/Km for the reactions of whole substrates catalyzed by 6-phosphogluconate dehydrogenase, glucose 6-phosphate dehydrogenase, and glucose 6-phosphate isomerase are reduced by 11-13 kcal/mol by interactions between the protein and the substrate phosphodianion. Between 4 and 6 kcal/mol of this dianion binding energy is expressed at the transition state for phosphite dianion activation of the respective enzyme-catalyzed reactions of truncated substrates d-xylonate or d-xylose. These and earlier results from studies on ß-phosphoglucomutase, triosephosphate isomerase, and glycerol 3-phosphate dehydrogenase define a cluster of six enzymes that catalyze reactions in glycolysis or of glycolytic intermediates, and which utilize substrate dianion binding energy for enzyme activation. Dianion-driven conformational changes, which convert flexible open proteins to tight protein cages for the phosphorylated substrate, have been thoroughly documented for five of these six enzymes. The clustering of metabolic enzymes which couple phosphodianion-driven conformational changes to enzyme activation suggests that this catalytic motif has been widely propagated in the proteome.