D-serine is a neuromodulator that co-activates NMDA glutamatergic receptors. It is produced in several cerebral regions and it is synthesized by serine racemase (SR), a neuronal pyridoxal 5’-phosphate (PLP)-dependent homodimeric enzyme that catalyzes the reversible racemization of L-serine to D-serine and the irreversible deamination of L- and D-serine to pyruvate and ammonia. D-serine has been demonstrated to be involved in amyotrophic lateral sclerosis (ALS) pathogenesis. Indeed, the deletion of the gene encoding SR slowed down the disease progression in ALS mouse models and protected against cerebral ischemia and excitotoxicity. It derives that SR is an interesting target for the development of innovative ALS treatments.<br/>It was found that SR is regulated by S-nitrosylation at Cys113, with a 7-fold reduction of the enzyme activity. To get insights into the mechanism of regulation, we investigated the correlation between S-nitrosylation and the SR conformations stabilized by ATP, a positive allosteric effector of SR, and glycine, a physiological inhibitor of SR. By integrating experimental and theoretical approaches, we demonstrated that S-nitrosylation-mediated negative regulation occurs through a specific molecular mechanism. Indeed, S-nitrosylation stabilizes an open, less-active conformation of the enzyme. The reaction of hSR with either NO or nitroso donors is conformation-dependent and occurs only in the conformation stabilized by ATP, in which the e-amino group of Lys114 acts as a base towards the thiol group of Cys113. In the closed conformation stabilized by glycine the side chain of Lys114 moves away from that of Cys113, while the carboxyl side-chain group of Asp318 moves significantly closer, increasing the thiol pK<sub>a</sub> and preventing the reaction. We conclude that ATP binding, glycine binding, and S-nitrosylation constitute a three-way regulation mechanism for the tight control of hSR activity.

S-nitrosylation and glycine control the activity of human serine racemase through an allosteric interplay

ACS Fall 2021

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