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. - 288:9(2021), pp. 3034-3054. [10.1111/febs.15645]

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

Marchesani F.;Michielon A.;Campanini B.;Faggiano S.;Bettati S.;Mozzarelli A.;Spyrakis F.
;
Bruno S.
2021-01-01

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 H2O2-mediated oxidation, with loss of enzyme activity, a reaction also dependent on hSR conformation.
2021
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. - 288:9(2021), pp. 3034-3054. [10.1111/febs.15645]
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11381/2931938
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 10
  • ???jsp.display-item.citation.isi??? 10
social impact