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

Serine racemase is the enzyme responsible for the synthesis of the neuromodulator D‐serine in neurons. ATP allosterically activates the enzyme, whereas S‐nitrosylation at a specific cysteine residue inhibits it. Here, we show that S‐nitrosylation occurs only in the presence of ATP and produces a conformational rearrangement toward a more open and less active structure. Ligand binding at the active site prevents nitrosylation. 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 H2O2‐mediated oxidation, with loss of enzyme activity, a reaction also dependent on hSR conformation.