Human serine racemase structure/activity relationship studies provide mechanistic insight and point to position 84 as a hot spot for -elimination function

There is currently great interest in human serine racemase, the enzyme responsible for producing the NMDA co-agonist d-serine. Reported correlation of d-serine levels with disorders including Alzheimer's disease, ALS, and ischemic brain damage (elevated d-serine) and schizophrenia (reduced d-serine) has further piqued this interest. Reported here is a structure/activity relationship study of position Ser(84), the putative re-face base. In the most extreme case of functional reprogramming, the S84D mutant displays a dramatic reversal of -elimination substrate specificity in favor of l-serine over the normally preferred l-serine-O-sulfate (approximate to 1200-fold change in k(cat)/K-m ratios) and l (l-THA; approximate to 5000-fold change in k(cat)/K-m ratios) alternative substrates. On the other hand, the S84T (which performs l-Ser racemization activity), S84A (good k(cat) but high K-m for l-THA elimination), and S84N mutants (nearly WT efficiency for l-Ser elimination) displayed intermediate activity, all showing a preference for the anionic substrates, but generally attenuated compared with the native enzyme. Inhibition studies with l-erythro--hydroxyaspartate follow this trend, with both WT serine racemase and the S84N mutant being competitively inhibited, with K-i = 31 +/- 1.5 m and 1.5 +/- 0.1 mm, respectively, and the S84D being inert to inhibition. Computational modeling pointed to a key role for residue Arg-135 in binding and properly positioning the l-THA and l-serine-O-sulfate substrates and the l-erythro--hydroxyaspartate inhibitor. Examination of available sequence data suggests that Arg-135 may have originated for l-THA-like -elimination function in earlier evolutionary variants, and examination of available structural data suggests that a Ser(84)-H2O-Lys(114) hydrogen-bonding network in human serine racemase lowers the pK(a) of the Ser(84)re-face base.