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.
The allosteric interplay between S-nitrosylation and glycine binding controls the activity of human serine racemase / Marchesani, F.; Gianquinto, E.; Autiero, I.; Michielon, A.; Campanini, B.; Faggiano, S.; Bettati, S.; Mozzarelli, A.; Spyrakis, F.; Bruno, S.. - In: THE FEBS JOURNAL. - ISSN 1742-464X. - (2020). [10.1111/febs.15645]
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