Substrate recognition and activation mechanism of D-amino acid oxidase: A study using substrate analogs

We investigated the mechanism of recognition and activation of substrate by D-amino acid oxidase (DAO) by thermodynamical and spectrophotometric metho ds using zwitterionic ligands [N-methylisonicotinate (NMIN), trigonelline, and homarine] and monoanionic ligands as model compounds of the substrate a nd the product. In terms of the charge within the substrate D-amino acid, m onoanionic (e.g., benzoate), zwitterionic (e.g., NMIN), and dianionic (e.g. , terephthalate) ligands are thought to be good models for neutral, basic, and acidic amino acids, respectively, because when a substrate binds to DAO , as previously reported, the alpha-ammonium group (-NH3+) probably loses a proton to become neutral (-NH2) before the oxidation. Zwitterionic ligands can also be good model compounds of product in the purple complex (the com plex of reduced DAO with the product imino acid), because the imino nitroge n of the imino acid is in a protonated cationic form. We also discuss elect rostatic interaction, steric effect, and charge-transfer interaction as fac tors which affect the affinity of substrate/ligand for DAO. Monoanionic lig ands have high affinity for neutral forms of oxidized and semiquinoid DAO, while zwitterionic ligands have high affinity for anionic forms of oxidized , semiquinoid, and reduced DAO; this difference was explained by the electr ostatic interaction in the active site. The low affinity of homarine (N-met hylpicolinate) for oxidized DAO, as in the case of o-methylbenzoate, is due to steric hindrance: one of the ortho carbons of benzoate is near the phen ol carbons of Tyr228 and the other ortho carbon is near the carbonyl oxygen of Gly313. The correlation of the affinity of meta- and para-substituted b enzoates for oxidized DAO with their Hammet's sigma values are explained by the HOMO-LUMO interaction between the phenol group of Tyr224 and the benze ne ring of benzoate derivative. The pK(a) of neutral flavin [N(3)-H of oxid ized flavin, N(5)-H of semiquinoid flavin, and N(1)-H of reduced flavin] de creases by its binding to the apoenzyme. The magnitude of the decrement is oxidized flavin < semiquinoid flavin < reduced flavin. The largest factor i n the substantially low pK(a) of reduced flavin in DAO is probably the ster ic hindrance between the hydrogen atom of H-N(1)(flavin) and the hydrogen a tom of H-N of Gly315, which becomes significant when a hydrogen is bound to N(1) of flavin.