Human serine racemase: regulation by divalent metals, NADH and S-nitrosylation

Human serine racemase (hSR) is a dimeric pyridoxal 5’ phosphate-dependent enzyme involved in the synthesis and metabolism of D-serine, the obligatory co-agonist of the NMDA receptors for glutamate. My PhD work focused on the characterization of some of its allosteric effectors. Divalent cations - Mg2+ and Ca2+ - and ATP both increase enzyme activity. By using analytical gel filtration chromatography, we also found that they shift the quaternary equilibrium towards a tetramer. NADH was also characterized and was found to act as a partial inhibitor, with the 1,4 dihydronicotinamidic ring as structural determinant. The NADH binding site was identified by molecular docking at the dimer interface, close to the ATP binding site. N-substituted 1,4 dihydronicotinamidic derivatives 1-methyl-1,4-dihydronicotinamide and -1,4-dihydronicotinamide mononucleotide were also found to be active, opening the way for the design of allosteric inhibitors. hSR was discovered to be S-nitrosylated at three cysteine residues (Cys113, Cys128 and Cys269), resulting in a biphasic time-dependent inhibition, with the fast phase associated to the nitrosylation of Cys113, as confirmed by the characterization of the C113S mutant. The affinity of nitrosylated hSR for ATP and active site ligands, as glycine or malonate, were found to be comparable to un-modified serine racemase. In the presence of active-site ligands, inhibition by S-nitrosylation was markedly slowed down, suggesting that S-nitrosylation is dependent on the conformational equilibrium. Site-directed mutagenesis of the residues adjacent to Cys113 helped elucidate the structural bases of hSR S-nitrosylation.