Biochemical characterization of human serine racemase: substrates, inhibitors and allosteric effectors

In recent years, many neurodegenerative diseases have been correlated to the dysfunction of N-methyl-D-aspartate receptors (NMDARs)-mediated neurotransmission. Drugs that directly affect the activity of the receptors evoke strong responses and typically show severe side effects. In this framework, serine racemase (SR) †" the enzyme responsible for D-serine production in the brain †" was suggested as an alternative therapeutic target. The detailed characterization of human SR (hSR) is currently rather limited, with a few published biochemical studies that provides a limited insight into the understanding of its regulation mechanisms. This work was aimed at achieving a more complete characterization of hSR dynamics, function and regulatory properties. After improving the protein purification yield and stability, which had so far limited the biochemical characterization of hSR, the dependence of L- and D-serine ?-elimination and L-serine racemization activities on ATP concentration was characterized and found to be strongly cooperative. ATP binding to the holo-enzyme, monitored by the fluorescence changes of the coenzyme, was also determined to be cooperative. The well-known active site ligands glycine and malonate increase the hSR affinity for ATP and, conversely, ATP increases the non-cooperative glycine and malonate binding. These results indicated a cross-talk between allosteric and active sites, leading to the stabilization of two alternative protein conformations endowed with significantly different microscopic dissociation constants for ATP , as estimated by applying the Monod, Wyman and Changeux model. To further investigate this allosteric communication, we probed the active site accessibility by quenching of the coenzyme fluorescence in the absence and presence of ATP. We found that ATP stabilized a closed conformation of the external aldimine Schiff base, suggesting a possible mechanism for ATP-induced hSR activation. We also investigated the effect of halides on hSR activity and we found that it is affected in the order fluoride>chloride>bromide. On the contrary, iodide elicited a complete inhibition, accompanied by a modulation of the tautomeric equilibrium of the internal aldimine. Finally, we applied two different approaches for the search of new hSR competitive inhibitors. With the first approach, we developed a new series of compounds based on cyclopropane scaffold and on malonate core modifications. With the second approach, a library of compound was screened against hSR structure. The positive molecules were tested with in vitro analysis. Our results can provide useful indications for the development of novel SR inhibitors.