TY - JOUR
T1 - Human serine racemase structure/activity relationship studies provide mechanistic insight and point to position 84 as a hot spot for β-elimination function
AU - Nelson, David L.
AU - Applegate, Greg A.
AU - Beio, Matthew L.
AU - Graham, Danielle L.
AU - Berkowitz, David B.
N1 - Publisher Copyright:
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2017/8/25
Y1 - 2017/8/25
N2 - 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 Lserine-O-sulfate (~1200-fold change in kcat/Km ratios) and L (L-THA; ~5000-fold change in kcat/Km ratios) alternative substrates. On the other hand, the S84T (which performs L-Ser racemization activity), S84A (good kcat but highKm for L-THAelimination), 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 Ki= 31±1.5μM and 1.5 ± 0.1μM, 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-likeβ-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 pKa of the Ser84re-face base.
AB - 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 Lserine-O-sulfate (~1200-fold change in kcat/Km ratios) and L (L-THA; ~5000-fold change in kcat/Km ratios) alternative substrates. On the other hand, the S84T (which performs L-Ser racemization activity), S84A (good kcat but highKm for L-THAelimination), 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 Ki= 31±1.5μM and 1.5 ± 0.1μM, 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-likeβ-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 pKa of the Ser84re-face base.
UR - http://www.scopus.com/inward/record.url?scp=85028409585&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85028409585&partnerID=8YFLogxK
U2 - 10.1074/jbc.M117.777904
DO - 10.1074/jbc.M117.777904
M3 - Article
C2 - 28696262
AN - SCOPUS:85028409585
SN - 0021-9258
VL - 292
SP - 13986
EP - 14002
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 34
ER -