The allosteric interplay between S-nitrosylation and glycine binding controls the activity of human serine racemase

FEBS J. 2021 May;288(9):3034-3054. doi: 10.1111/febs.15645. Epub 2020 Dec 21.

Abstract

Human serine racemase (hSR) catalyzes the biosynthesis of D-serine, an obligatory co-agonist of the NMDA receptors. It was previously found that the reversible S-nitrosylation of Cys113 reduces hSR activity. Here, we show by site-directed mutagenesis, fluorescence spectroscopy, mass spectrometry, and molecular dynamics that S-nitrosylation stabilizes an open, less-active conformation of the enzyme. The reaction of hSR with either NO or nitroso donors is conformation-dependent and occurs only in the conformation stabilized by the allosteric effector ATP, in which the ε-amino group of Lys114 acts as a base toward the thiol group of Cys113. In the closed conformation stabilized by glycine-an active-site ligand of hSR-the side chain of Lys114 moves away from that of Cys113, while the carboxyl side-chain group of Asp318 moves significantly closer, increasing the thiol pKa and preventing the reaction. We conclude that ATP binding, glycine binding, and S-nitrosylation constitute a three-way regulation mechanism for the tight control of hSR activity. We also show that Cys113 undergoes H2 O2 -mediated oxidation, with loss of enzyme activity, a reaction also dependent on hSR conformation.

Keywords: D-serine; NMDA receptors; allosteric modulation; fluorescence spectroscopy; glycine; molecular dynamics; nitrosylation; pyridoxal phosphate; serine racemase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allosteric Regulation / genetics*
  • Binding Sites
  • Catalytic Domain / genetics
  • Glycine / genetics
  • Humans
  • Kinetics
  • Oxidation-Reduction
  • Protein Conformation*
  • Racemases and Epimerases / chemistry
  • Racemases and Epimerases / genetics
  • Racemases and Epimerases / ultrastructure*

Substances

  • Racemases and Epimerases
  • serine racemase
  • Glycine