biostudies-literatureNelson DLAmerican Heart AssociationNCRR NIH HHSNational Institutes of Health13986-14002https://www.ebi.ac.uk/biostudies/studies/S-EPMC5572919biostudies-literatureUnknown292(34)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 <i>re</i>-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-<i>O</i>-sulfate (∼1200-fold change in <i>k</i>_cat/<i>K_m</i> ratios) and l (l-THA; ∼5000-fold change in <i>k</i>_cat/<i>K_m</i> ratios) alternative substrates. On the other hand, the S84T (which performs l-Ser racemization activity), S84A (good <i>k</i>_cat but high <i>K_m</i> 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-<i>erythro</i>-β-hydroxyaspartate follow this trend, with both WT serine racemase and the S84N mutant being competitively inhibited, with <i>K_i</i> = 31 ± 1.5 μm 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-<i>O</i>-sulfate substrates and the l-<i>erythro</i>-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 p<i>K_a</i> of the Ser⁸⁴<i>re</i>-face base.The Journal of biological chemistryHuman serine racemase structure/activity relationship studies provide mechanistic insight and point to position 84 as a hot spot for β-elimination function.PMC557291916GRNT313400012RR016544C06 RR016544Graham DLNelson DLBerkowitz DBBeio MLApplegate GAfalseHuman serine racemase structure/activity relationship studies provide mechanistic insight and point to position 84 as a hot spot for β-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 Ser⁸⁴, the putative <i>re</i>-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-<i>O</i>-sulfate (∼1200-fold change in <i>k</i>_cat/<i>K_m</i> ratios) and l (l-THA; ∼5000-fold change in <i>k</i>_cat/<i>K_m</i> ratios) alternative substrates. On the other hand, the S84T (which performs l-Ser racemization activity), S84A (good <i>k</i>_cat but high <i>K_m</i> 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-<i>erythro</i>-β-hydroxyaspartate follow this trend, with both WT serine racemase and the S84N mutant being competitively inhibited, with <i>K_i</i> = 31 ± 1.5 μm 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-<i>O</i>-sulfate substrates and the l-<i>erythro</i>-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 p<i>K_a</i> of the Ser⁸⁴<i>re</i>-face base.2017-01-01T00:00:00Z2017 Aug2025-04-26T01:24:15.321Z2019-03-26T23:52:10ZS-EPMC55729192869626210.1074/jbc.m117.77790410.1074/jbc.M117.777904