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

Authors

David L. Nelson, University of Nebraska - Lincoln
Greg A. Applegate, University of Nebraska - Lincoln
Matthew L. Beio, University of Nebraska-LincolnFollow
Danielle L. Graham, University of Nebraska - LincolnFollow
David B. Berkowitz, University of Nebraska - LincolnFollow

Document Type

Article

Date of this Version

2017

Citation

J. Biol. Chem. (2017) 292(34) 13986–14002

Comments

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI 10.1074/jbc.M117.777904

Abstract

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 Ser⁸⁴, the putative re-face base. In the most extreme case of functional reprogramming, the S84D mutant displays a dramatic reversal of β-elimination substrate specificity in favor of L-serine over the normally preferred L-serine-O-sulfate (~1200-fold change in k_cat/K_m ratios) and L (L-THA; ~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-β-hydroxyaspartate follow this trend, with both WT serine racemase and the S84N mutant being competitively inhibited, with K_i = 31 ± 1.5 μM and 1.5 ± 0.1mM, 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-β-hydroxyaspartate inhibitor. Examination of available sequence data suggests that Arg-135 may have originated for L-THA-like-β-elimination function in earlier evolutionary variants, and examination of available structural data suggests that a Ser⁸⁴-H₂O-Lys¹¹⁴ hydrogen-bonding network in human serine racemase lowers the pK_a of the Ser⁸⁴ re-face base.