Ana Ríos Rodríguez and Juan Crugeiras
Rate constants for deprotonation of carbon acids in D2O can be determined by using 1H NMR spectroscopy to monitor the transfer of deuterium from solvent to the carbon acid. These rate constants can then be combined with known rate-equilibrium relationships for proton transfer at carbon to estimate (pKa)CH values for these carbon acids. We have applied this methodology to study the kinetic and thermodynamic stability of enolates of amino acids and their derivatives in aqueous solution (JACS 1997, 119, 8375; JACS 2000, 122, 9373; JACS 2002, 124, 8251). This work has provided the first reliable values of the carbon acidity in water of amino acids. These (pKa)CH values are essential to characterize the chemical reactivity of these biological compounds.
These results are relevant to the mechanism of catalysis of amino acid racemases that do not require a cofactor for their catalytic activity. Our study suggests two related roles for the enzyme catalyst in optimizing the strong electrostatic stabilization of these enolates due to interactions between the positive ammonium group and the negative charge of the enolate oxygen. (a) Amino acid racemases bind the most abundant form of amino acids at physiological pH, the zwitterion, and activate this species toward enolization by either preequilibrium protonation of the carboxylate or by general acid catalysis at the carbonyl group. (b) The enzyme active site provides a medium of low dielectric constant that maximizes stabilization of the zwitterionic enolate intermediate by intramolecular electrostatic interactions.
