Magnesium and calcium ions differentially affect human serine racemase activity and modulate its quaternary equilibrium toward a tetrameric form

•Mg2 + activates allosterically serine racemase in competition with Ca2 +.•The ATP-Ca2 + complex produces half the activation brought about by the ATP-Mg2 + complex.•Ca2 + and Mg2 + stabilize a tetrameric form of the enzyme.•ATP, in complex with Ca2 + or Mg2 +, further shifts the quaternary equilibrium toward the tetrameric form.•Ca2 + and Mg2 + increase the thermal stability of the enzyme.

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.

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