ATP binding to human serine racemase is cooperative and modulated by glycine


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Abstract

The N-methyl d-aspartate (NMDA) receptors play a key role in excitatory neurotransmission, and control learning, memory and synaptic plasticity. Their activity is modulated by the agonist glutamate and by the co-agonists d-serine and glycine. In the human brain, d-serine is synthesized from l-serine by the dimeric pyridoxal 5′-phosphate-dependent enzyme serine racemase, which also degrades l- and d-serine to pyruvate and ammonia. The dependence of l- and d-serine β-elimination and l-serine racemization activities on ATP concentration was characterized, and was found to be strongly cooperative, with Hill coefficients close to 2 and apparent ATP dissociation constants ranging from 0.22 to 0.41 mm. ATP binding to the holo-enzyme, monitored by the fluorescence changes of the coenzyme, was also determined to be cooperative, with an apparent dissociation constant of 0.24 mm. Glycine, an active-site ligand, increased the serine racemase affinity for ATP by ˜ 22-fold, abolishing cooperativity. Conversely, ATP increased the non-cooperative glycine binding15-fold. These results indicate cross-talk between allosteric and active sites, leading to the stabilization of two alternative protein conformations with ATP affinities of ˜ 10 μM and 1.8 mm, as evaluated within the Monod, Wyman and Changeux model. Therefore, intracellular ATP and glycine control d-serine homeostasis, and, indirectly, NMDA receptor activity. Because hyper- and hypo-activation of NMDA receptors are associated with neuropathologies, the development of allosteric drugs modulating serine racemase activity is a promising therapeutic strategy.NMDA receptors are modulated by the co-agonists glycine and d-serine, the latter being synthesized by serine racemase. Binding of the allosteric ligand ATP to the enzyme is strongly cooperative. Glycine, a substrate analog, increases ATP affinity and abolishes cooperativity. These results support the notion of a crosstalk between allosteric and active sites and the existence of alternative protein conformations.

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