Serine hydroxymethyltransferase: mechanism of the racemization and transamination of D- and L-alanine

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Abstract
The reaction specificity and sterochemical control of Escherichia coli serine hydroxymethyltransferase were investigated with D- and L-alanine as substrates. An active-site H228N mutant enzymes binds both D- and L-alanine with Kd values of 5 mM as compared to 30 and 10 mM, respectively, for the wild-type enzyme. Both wild-type and H228N enzymes form quinonoid complexes absorbing at 505 mm by catalyzing the loss of the .alpha.-proton from both D- and L-alanine. Racemization and transamination reactions were observed to occur with both alanine isomers as substrates. The relative rates of these reactions are quinonoid formation > .alpha.-proton solvent exchange > racemization > transamination. The observation that the rate of quinoid formation with either alanine isomer is an order of magnitude faster than solvent exchange suggests that the .alpha.-protons from both D- and L-alanine are transferred to base(s) on the enzyme. The rate of racemization is 2 orders of magnitude slower than the formation of the quinonoid complexes. This latter difference in rate suggests that the quinonoid complexes formed from D- and L-alanine are not identical. The difference in structure of the two quinoid complexes is proposed to be the active-site location of the .alpha.-protons lost from the two alanine isomers, rather than two orientations of the pyridoxal phosphate ring. The results are consistent with a two-base mechanism for racemization.