title: Magnesium and calcium ions differentially affect human serine racemase activity and modulate its quaternary equilibrium toward a tetrameric form.
abstract: Serine racemase is the pyridoxal 5'-phosphate dependent enzyme that catalyzes both production and catabolism of d-serine, a co-agonist of the NMDA glutamate receptors. Mg2+, or, alternatively, Ca2+, activate human serine racemase by binding both at a specific site and - as ATP-metal complexes - at a distinct ATP binding site. We show that Mg2+and Ca2+bind at the metal binding site with a 4.5-fold difference in affinity, producing a similar thermal stabilization and partially shifting the dimer-tetramer equilibrium in favour of the latter. The ATP-Ca2+complex produces a 2-fold lower maximal activation in comparison to the ATP-Mg2+complex and exhibits a 3-fold higher EC50. The co-presence of ATP and metals further stabilizes the tetramer. In consideration of the cellular concentrations of Mg2+and Ca2+, even taking into account the fluctuations of the latter, these results point to Mg2+as the sole physiologically relevant ligand both at the metal binding site and at the ATP binding site. The stabilization of the tetramer by both metals and ATP-metal complexes suggests a quaternary activation mechanism mediated by 5'-phosphonucleotides similar to that observed in the distantly related prokaryotic threonine deaminases. This allosteric mechanism has never been observed before in mammalian fold type II pyridoxal 5'-phosphate dependent enzymes.
authors.affiliation: Department of Pharmacy, University of Parma, Italy. Electronic address: stefano.bruno@unipr.it.
authors.forename: Stefano
authors.initials: S
authors.lastname: Bruno
authors.affiliation: Department of Pharmacy, University of Parma, Italy.
authors.forename: Marilena
authors.initials: M
authors.lastname: Margiotta
authors.affiliation: Department of Pharmacy, University of Parma, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Marchesani
authors.affiliation: Interdepartment Center SITEIA.PARMA, University of Parma, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Paredi
authors.affiliation: Department of Pharmacy, University of Parma, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Orlandi
authors.affiliation: Department of Pharmacy, University of Parma, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Faggiano
authors.affiliation: Department of Neurosciences, University of Parma, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Ronda
authors.affiliation: Department of Pharmacy, University of Parma, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Campanini
authors.affiliation: Department of Pharmacy, University of Parma, Italy; Institute of Biophysics, CNR, Pisa, Italy; National Institute of Biostructures and Biomolecules, Rome, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Mozzarelli
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D006801:Humans; D008274:Magnesium / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D019998:Racemases and Epimerases / Q000737:chemistry*; D000099300:Serine Racemase
chemical_list: D000255:Adenosine Triphosphate; D002118:Calcium; D008274:Magnesium; D019998:Racemases and Epimerases; D000099300:Serine Racemase
keywords: Allosteric regulation; Enzyme regulation; Metal-protein interaction; Metalloproteins; Serine racemase