Regulation of human serine racemase activity and dynamics by halides, ATP and malonate

Abstract

D-Serine is a non-proteinogenic amino acid that acts as a co-agonist of the NMDA receptors in the central nervous system. D-Serine is produced by human serine racemase (hSR), a homodimeric pyridoxal 5'-phosphate (PLP)-dependent enzyme that also catalyzes the physiologically relevant β-elimination of both L- and D-serine to pyruvate and ammonia. After improving the protein purification yield and stability, which had so far limited the biochemical characterization of hSR, we found that the catalytic activity is affected by halides, 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. We also investigated the reciprocal effects of ATP and malonate, an inhibitor that reversibly binds at the active site, 20 Å away from the ATP-binding site. ATP increased ninefold the affinity of hSR for malonate and malonate increased 100-fold that of ATP, confirming an allosteric interaction between the two binding sites. 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 stabilizes a closed conformation of the external aldimine Schiff base, suggesting a possible mechanism for ATP-induced hSR activation.

Glutamine 89 is a key residue in the allosteric modulation of human serine racemase activity by ATP

“…However, plant SRs represent a distinct group in eukaryotic serine racemases 9 and are thus likely to be all insensitive to nucleotide binding. Interestingly, the communication between active site and the ATP binding site is bidirectional, with the affinity for ATP increasing in the presence of active site ligands such as glycine and malonate 18 , 23 . The occupation of the active site by substrates and/or inhibitors leads to conformational changes in the small domain, which rotates with respect to the large domain to restrain accessibility to the active site 14 , 24 .…”

The Energy Landscape of Human Serine Racemase

“…As shown above, the comparison of the structure of SpSR in the absence and presence of the ATP analog AMP-PCP (PDB 1V71 and 1WTC) revealed a change in the relative orientation of the major and minor domain (Goto et al, 2009), resulting in a wider back groove. Moreover, in the presence of ATP and either glycine or malonate, hSR active site appears less accessible than in the absence of ATP, as demonstrated by PLP fluorescence quenching (Marchetti et al, 2015). Moreover, by monitoring PLP fluorescence in the absence and presence of ATP, it was demonstrated that ATP binding to hSR promotes a decrease in the polarity of the active site, diminishing its accessibility (Marchetti et al, 2013).…”

Crystal structure of maize serine racemase with pyridoxal 5′-phosphate

“…The structure of fission yeast SR in complex with the ATP analogue AMP-PCP shows that 14 residues are involved in ATP binding (Goto et al, 2009). The racemase activity of human SR is increased by ATP (Marchetti et al, 2013(Marchetti et al, , 2015.…”