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 alpha-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 alpha-imino carbon increases from 7.5 x 10(2) to 3.8 x 10(5) to 3.0 x 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(a)s for deprotonation of the alpha-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 x 10(-3) s(-1) for deprotonation of the alpha-imino carbon of this cofactor-glycine adduct (pK(a) = 17 by HPO42- with k(cat)/K-m = 4 x 10(5) M-1 s(-1) for catalysis of amino-acid racemization by alanine racemase shows that the enzyme causes a ca 2 x 10(8)-fold acceleration of the rate of deprotonation the alpha-imino carbon. This corresponds to about one-half of the burden borne by alanine racemase in catalysis of deprotonation of alanine.