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

David L. Nelson, Greg A. Applegate, Matthew L. Beio, Danielle L. Graham, David B. Berkowitz
2017 Journal of Biological Chemistry  
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
more » ... 4D mutant displays a dramatic reversal of ␤-elimination substrate specificity in favor of L-serine over the normally preferred Lserine-O-sulfate (ϳ1200-fold change in k cat /K m ratios) and L (L-THA; ϳ5000-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 -H 2 O-Lys 114 hydrogen-bonding network in human serine racemase lowers the pK a of the Ser 84 re-face base. Figure 12 . Phylogenetic tree of various fold type II eliminases. Enzymes annotated as SR are marked with blue dots; enzymes annotated as having CBS activity are given lavender triangles.
doi:10.1074/jbc.m117.777904 pmid:28696262 pmcid:PMC5572919 fatcat:bgvs6lyoenafxdonadcmphf2zi