Covalent Catalysis by Pyridoxal: Evaluation of the Effect of the Cofactor on the Carbon Acidity of Glycine

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Abstract
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 kHO for hydroxide ion-catalyzed deprotonation of the α-imino carbon increases from 7.5 × 102 to 3.8 × 105 to 3.0 × 107 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 pKas 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 pKa = 17. A comparison of kB = 1.7 × 10-3 s-1 for deprotonation of the α-imino carbon of this cofactor-glycine adduct (pKa = 17 by HPO42- with kcat/Km = 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.

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