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

Research output: Contribution to journalArticle

5 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)13986-14002
Number of pages17
JournalJournal of Biological Chemistry
Volume292
Issue number34
DOIs
StatePublished - Aug 25 2017

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Structure-Activity Relationship
Serine
Tacrine
Substrates
N-Methylaspartate
Enzymes
Hydrogen Bonding
Substrate Specificity
Sulfates
Brain
Schizophrenia
Hydrogen bonds
Alzheimer Disease
serine racemase
Efficiency
serine containing aminolipid
Inhibition (Psychology)

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Human serine racemase structure/activity relationship studies provide mechanistic insight and point to position 84 as a hot spot for β-elimination function. / Nelson, David L.; Applegate, Greg A.; Beio, Matthew L.; Graham, Danielle L.; Berkowitz, David B.

In: Journal of Biological Chemistry, Vol. 292, No. 34, 25.08.2017, p. 13986-14002.

Research output: Contribution to journalArticle

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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 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.",
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AU - Graham, Danielle L.

AU - Berkowitz, David B

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