The ratio of the second-order rate constants for reduction of dihydroxyacetone phosphate (DHAP) and of the neutral truncated substrate glycolaldehyde (GLY) by glycerol 3-phosphate dehydrogenase (NAD + , GPDH) saturated by NADH is (1.0×10 6 M −1 s −1 )/(8.7×10 −3 M −1 s −1 ) = 1.1×10 8 , which was used to calculate an intrinsic phosphate binding energy of ≤ −11.0 kcal/mol. Phosphite dianion binds very weakly to GPDH (K d > 0.1 M), but the bound dianion strongly activates GLY towards enzymecatalyzed reduction by NADH. Thus, the large intrinsic phosphite binding energy is expressed only at the transition state for the GPDH-catalyzed reaction. The ratio of rate constants for the phosphite-activated and the unactivated GPDH-catalyzed reduction of GLY by NADH is (4300 M −2 s −1 )/(8.7×10 −3 M −1 s −1 ) = 5×10 5 M −1 , which was used to calculate an intrinsic phosphite binding energy of −7.7 kcal/mol for the association of phosphite dianion with the transition state complex for the GPDH-catalyzed reduction of GLY. Phosphite dianion has now been shown to activate bound substrates for enzyme-catalyzed proton transfer, decarboxylation, hydride transfer and phosphoryl transfer reactions. Structural data provides strong evidence that enzymic activation by the binding of phosphite dianion occurs at a modular active site featuring: (1) a binding pocket complementary to the reactive substrate fragment, and which contains all the active site residues needed to catalyze the reaction of the substrate piece or of the whole substrate; (2) a phosphate/phosphite dianion binding pocket that is completed by the movement of flexible protein loop(s) to surround the nonreacting oxydianion. We propose that loop motion and associated protein conformational changes that accompany the binding of phosphite dianion and/or phosphodianion substrates leads to encapsulation of the substrate and/or its pieces in the protein interior, and to placement of the active site residues in positions where they provide optimal stabilization of the transition state for the catalyzed reaction.Enzymes are protein catalysts that effect much larger rate accelerations than those observed for small molecule catalysts. The larger rate accelerations for enzymes are a consequence of the greater binding affinities of enzymes for their reaction transition states (1). Enzyme catalysis is so efficient that the release of products would be intolerably slow if they were to bind with the same affinity as the transition state. For example, the ca. 30 kcal/mol transition state intrinsic binding energy estimated for the enzymatic decarboxylation of orotidine 5′-monophosphate (OMP) 1 catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) (2) is even larger than the 20 kcal/mol binding energy associated with the effectively irreversible binding of biotin to avidin (3). Consequently, in order to avoid this free energy "trap", enzymes generally bind their substrates/products much more weakly than the reaction transition state.

Primary product isotope effects (PIEs) on L + -and carboxylic acid-catalyzed protonation of ringsubstituted α-methoxystyrenes (X-1) to form oxocarbenium ions X-2 + in 50/50 (v/v) HOH/DOD were calculated from the yields of the α-CH 3 and α-CH 2 D labeled ketone products, determined by 1 H NMR. A plot of PIE against reaction driving force shows a maximum PIE of 8. These parameters are consistent with reaction of the hydron over an energy barrier. There is no evidence for quantum mechanical tunneling of the hydron through the barrier. These PIEs suggest that the transferred hydron at the transition state lies roughly equidistant between the acid donor and base acceptor, and contrast with the recently published Brønsted parameters [Richard, J. P.; Williams, K. B. J. Am. Chem. Soc. 2007, 129, 6952-6961], which are consistent with a product like transition state. An explanation for these seemingly contradictory results is discussed.

We report a fast, simple and general method to determine primary kinetic isotope effects (KIEs) on proton transfer in hydroxylic solvents, and its application to obtain primary deuterium isotope effects on protonation of ring-substituted α-methoxystyrenes X-1 (Scheme 1) over a broad range of temperature and thermodynamic driving force. These data provide evidence that hydron transfer at X-1 proceeds by passing over a reaction barrier, as opposed to tunneling through the barrier; and, insight into the relationship between the primary kinetic isotope effect and the transition state structure.

To address the question of concerted versus a stepwise reaction mechanisms for the cyclization of the 2-hydroxypropyl aryl and alkyl RNA models (1a-k) promoted by dinuclear Zn(II) complex (4) at (s)spH 9.8 and 25 degrees C, the non-cleavable O-hydroxypropyl phenylphosphonate analogues 6a and 6b were subjected to the catalytic reaction in methanol. These phosphonates did not undergo isomerization in the study, the only observable methanolysis reaction being release of 1,2-propanediol and the formation of O-methyl phenylphosphonate. The observed first order rate constants for methanolysis promoted by 4 are k(obs)(6a) = (1.47 +/- 0.09) x 10(-4) s(-1) and k(obs)(6b) = (2.08 +/- 0.09) x 10(-6) s(-1), respectively. The rates of methanolysis of a series of O-aryl phenylphosphonates (8a-f) in the presence of increasing [4] were analyzed to provide binding constants, Kb, and the catalytic rate constant, kcat(max), for the unimolecular decomposition of the 8:4 Michaelis complex. A Brønsted plot of the log (k(cat)(max)) vs. sspKa(phenol) (acidity constant of the conjugate acid of the leaving group in methanol) was fitted to a linear regression of log kcat(max) = (-0.80 +/- 0.07)(s)spKa + (10.2 +/- 1.0) which includes the datum for 6a. The datum for 6b, which reacts approximately 70-fold slower, falls significantly below the linear correlation. The data provide additional evidence consistent with a concerted cyclization of RNA models 1a-k promoted by 4.

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