Covalent catalysis by pyridoxal: Evaluation of the effect of the cofactor on the carbon acidity of glycine

First-order rate constants for deprotonation of the α-imino carbon of the adduct between 5′-deoxypyridoxal (1) and glycine were determined as the rate constants for Claisen-type addition of glycine to 1 where deprotonation is rate determining for product formation. There is no significant deprotonation at pH 7.1 of the form of the 1-glycine iminium ion with the pyridine nitrogen in the basic form. The value of k_HO for hydroxide ion-catalyzed deprotonation of the α-imino carbon increases from 7.5 × 10² to 3.8 × 10⁵ to 3.0 × 10 ⁷ M^-1 s^-1, respectively, with protonation of the pyridine nitrogen, the phenoxide oxyanion, and the carboxylate anion of the 1-glycine iminium ion. There is a corresponding decrease in the pK_as for deprotonation of the α-imino carbon from 17 to 11 to 6. It is proposed that enzymes selectively bind and catalyze the reaction of the iminium ion with pK_a = 17. A comparison of k_B = 1.7 × 10 ^-3 s^-1 for deprotonation of the α-imino carbon of this cofactor-glycine adduct (pK_a = 17 by HPO₄ ^2- with k_cat/K_m = 4 × 10⁵ M^-1 s^-1 for catalysis of amino-acid racemization by alanine racemase shows that the enzyme causes a ca 2 × 10⁸-fold acceleration of the rate of deprotonation the α-imino carbon. This corresponds to about one-half of the burden borne by alanine racemase in catalysis of deprotonation of alanine.