We report second-order rate constants kDO (M-1 s-1) for exchange for deuterium of the C(2)-proton of a series of simple imidazolium cations to give the corresponding singlet imidazol-2-yl carbenes in D2O at 25 degrees C and I = 1.0 (KCl). Evidence is presented that the reverse protonation of imidazol-2-yl carbenes by solvent water is limited by solvent reorganization and occurs with a rate constant of kHOH = kreorg = 10(11) s-1. The data were used to calculate reliable carbon acid pK(a)s for ionization of imidazolium cations at C(2) to give the corresponding singlet imidazol-2-yl carbenes in water: pKa = 23.8 for the imidazolium cation, pKa = 23.0 for the 1,3-dimethylimidazolium cation, pKa = 21.6 for the 1,3-dimethylbenzimidazolium cation, and pKa = 21.2 for the 1,3-bis-((S)-1-phenylethyl)benzimidazolium cation. The data also provide the thermodynamic driving force for a 1,2-hydrogen shift at a singlet carbene: K12 = 5 x 10(16) for rearrangement of the parent imidazol-2-yl carbene to give neutral imidazole in water at 298 K, which corresponds to a favorable Gibbs free energy change of 23 kcal/mol. We present a simple rationale for the observed substituent effects on the thermodynamic stability of N-heterocyclic carbenes relative to a variety of neutral and cationic derivatives that emphasizes the importance of the choice of reference reaction when assessing the stability of N-heterocyclic carbenes.

More than 80% of the rate acceleration for enzymatic catalysis of the aldose-ketose isomerization of (R)-glyceraldehyde 3-phosphate (GAP) by triosephosphate isomerase (TIM) can be attributed to the phosphodianion group of GAP [Amyes, T. L., O'Donoghue, A. C., and Richard, J. P. (2001) J. Am. Chem. Soc. 123, 11325-11326]. We examine here the necessity of the covalent connection between the phosphodianion and triose sugar portions of the substrate by "carving up" GAP into the minimal neutral two-carbon sugar glycolaldehyde and phosphite dianion pieces. This "two-part substrate" preserves both the alpha-hydroxycarbonyl and oxydianion portions of GAP. TIM catalyzes proton transfer from glycolaldehyde in D2O, resulting in deuterium incorporation that can be monitored by 1H NMR spectroscopy, with kcat/Km = 0.26 M-1 s-1. Exogenous phosphite dianion results in a very large increase in the observed second-order rate constant (kcat/Km)obsd for turnover of glycolaldehyde, and the dependence of (kcat/Km)obsd on [HPO32-] exhibits saturation. The data give kcat/Km = 185 M-1 s-1 for turnover of glycolaldehyde by TIM that is saturated with phosphite dianion so that the separate binding of phosphite dianion to TIM results in a 700-fold acceleration of proton transfer from carbon. The binding of phosphite dianion to the free enzyme (Kd = 38 mM) is 700-fold weaker than its binding to the fleeting complex of TIM with the altered substrate in the transition state (Kd = 53 muM); the total intrinsic binding energy of phosphite dianion in the transition state is 5.8 kcal/mol. We propose a physical model for catalysis by TIM in which the intrinsic binding energy of the substrate phosphodianion group is utilized to drive closing of the "mobile loop" and a protein conformational change that leads to formation of an active site environment that is optimally organized for stabilization of the transition state for proton transfer from alpha-carbonyl carbon.

No abstract

The rate constants for deprotonation of ethyl acetate by 3-substituted quinuclidines are correlated by β ) 1.09 ( 0.05. The limits of k BH ) 2-5 × 10 9 M -1 s -1 for the encounter-limited reaction of the simple oxygen ester enolate with protonated quinuclidine (pK BH ) 11.5) were combined with k B ) 2.4 × 10 -5 M -1 s -1 for deprotonation of ethyl acetate by quinuclidine, to give pK a K) 25.6 ( 0.5 for ionization of ethyl acetate as a carbon acid in aqueous solution. A rate-equilibrium correlation for proton transfer from methyl and benzylic monocarbonyl compounds to hydroxide ion has been extended by 6 pK units in the thermodynamically unfavorable direction, and it is shown that the absence of curvature of this correlation is inconsistent with a constant Marcus intrinsic barrier for the enolization of simple carbonyl compounds.

We report that the binding of phosphite dianion to orotidine 5'-monophosphate decarboxylase (OMPDC) results in an 80 000-fold increase in kcat/Km for decarboxylation of the truncated substrate, 1-(beta-d-erythrofuranosyl)orotic acid (EO), which lacks a 5'-phosphodianion moiety. The intrinsic binding energy (IBE) of phosphite dianion in the transition state is 7.8 kcal/mol, which represents a very large fraction of the 11.8 kcal/mol IBE of the phosphodianion group of the natural substrate orotidine 5'-monophosphate (OMP). The data give kcat = 160 +/- 70 s-1 for turnover of EO in the active site of OMPDC containing phosphite dianion, which is significantly larger than kcat = 15 s-1 for turnover of OMP. Despite the weaker binding of the individual EO and HPO32- "parts" (KmKd = 0.014 M2) than of OMP (Km = 1.6 x 10-6 M), once bound, OMPDC provides a slightly greater stabilization of the transition state for reaction of the parts than of the whole substrate. Thus, the covalent connection between the reacting portion of the substrate and the nonreacting phosphodianion group is not necessary for efficient catalysis. This implies that a major role of the phosphodianion group of OMP is to provide binding interactions that are used to drive an enzyme conformational change, resulting in formation of an active site environment optimized for transition state stabilization.