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 formate dehydrogenase (FDH) for catalysis of hydride transfer from formate to NAD. The ADP-ribose fragment provides a >14 kcal/mol stabilization of the transition state for FDH-catalyzed hydride transfer from formate to NAD.

Phosphodianion Activation of Enzymes for Catalysis of Central Metabolic Reactions.

The activation barriers Δ for / 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 β-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.

-(Hydroxybenzyl)benzamide Derivatives: Aqueous pH-Dependent Kinetics and Mechanistic Implications for the Aqueous Reactivity of Carbinolamides.

The rate constants for the aqueous reaction, between pH 0 and 14, have been determined for a series of amide substituted -(hydroxybenzyl)benzamide derivatives, in HO, at 25 °C, = 1.0 M (KCl). The -(hydroxybenzyl)benzamide derivatives were found to react via three distinct mechanisms with the kinetically dominant mechanism being dependent on the pH of the reaction solution.

Determination of the pKa of cyclobutanone: Brønsted correlation of the general base-catalyzed enolization in aqueous solution and the effect of ring strain.

The induction of strain in carbocycles, thereby increasing the amount of s-character in the C-H bonds and the acidity of these protons, has been probed with regard to its effect on the rate constants for the enolization of cyclobutanone. The second-order rate constants for the general base-catalyzed enolization of cyclobutanone have been determined for a series of 3-substituted quinuclidine buffers in D(2)O at 25 °C, I = 1.0 M (KCl).

Amidates as leaving groups: structure/reactivity correlation of the hydroxide-dependent E1cB-like breakdown of carbinolamides in aqueous solution.

The kinetic study of the aqueous reaction, between pH 10 and 14, of eight N-(hydroxymethyl)benzamide derivatives in water at 25 degrees C, I = 1.0 M (KCl), has been performed. In all cases, the reaction proceeds via a specific-base-catalyzed deprotonation of the hydroxyl group followed by rate-limiting breakdown of the alkoxide to form aldehyde and amidate (E1cB-like).

Ring strain and its effect on the rate of the general-base catalyzed enolization of cyclobutanone.

[reaction: see text] The 3-quinuclidinone-catalyzed (pK(BH) = 7.5) enolization of cyclobutanone (1) in D(2)O at 25 degrees C, I = 1.0 (KCl) was followed by deuterium incorporation, which was determined by (1)H NMR.