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.

Keywords: Allosteric regulation; Enzyme regulation; Metal-protein interaction; Metalloproteins; Serine racemase.