title: 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.
authors.affiliation: Dipartimento di Bioscienze, Università di Parma, Parma, Italy.
authors.forename: Marialaura
authors.initials: M
authors.lastname: Marchetti
authors.forename: Stefano
authors.initials: S
authors.lastname: Bruno
authors.forename: Barbara
authors.initials: B
authors.lastname: Campanini
authors.forename: Stefano
authors.initials: S
authors.lastname: Bettati
authors.forename: Alessio
authors.initials: A
authors.lastname: Peracchi
authors.forename: Andrea
authors.initials: A
authors.lastname: Mozzarelli
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001965:Bromides / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D002712:Chlorides / Q000737:chemistry / Q000378:metabolism*; D005459:Fluorides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008314:Malonates / Q000737:chemistry / Q000378:metabolism*; D019998:Racemases and Epimerases / Q000737:chemistry / Q000378:metabolism*; D000099300:Serine Racemase
chemical_list: D000255:Adenosine Triphosphate; D001965:Bromides; D002712:Chlorides; D005459:Fluorides; D008314:Malonates; D019998:Racemases and Epimerases; D000099300:Serine Racemase; C030290:malonic acid