Proton release from flavoprotein D-amino acid oxidase on complexation with the zwitterionic ligand, trigonelline.
Trigonelline, i.e., N-methylnicotinate, which has a zwitterionic structure similar to a substrate D-amino acid, is a useful active site probe for D-amino acid oxidase (DAO). The affinity of trigonelline for DAO in the deprotonated state at the enzyme bound FAD 3-imino group is higher than in the neutral state, contrary to in the case of benzoate, which is a competitive inhibitor and is in a monoanionic form. The time course of the absorbance change was monitored for the binding of DAO with trigonelline by means of a stopped-flow technique. The reaction, on monitoring at 507 nm, was found to be biphasic at pH 8.3, with fast and slow phases. The dissociation of the 3-imino proton of the enzyme bound FAD was observed in the same time course as the slow phase. These results suggest that the positive charge of trigonelline exists near the 3-imino group of the enzyme bound FAD and interacts repulsively with the proton of the 3-imino group. The absorption spectra of the DAO-trigonelline complex at various pHs also support this hypothesis. In the catalysis of DAO, a similar mechanism may be involved, that is, the positive charge of a D-amino acid may interact repulsively with the 3-imino proton of the enzyme bound FAD, and this interaction may be important for the catalysis.
Nishina
Y
Y
Department of Physiology, Kumamoto University Medical School.
Sato
K
K
Shiga
K
K
D000470:Alkaloids / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D013053:Spectrophotometry; D013552:Swine
D000470:Alkaloids; D005182:Flavin-Adenine Dinucleotide; C009560:trigonelline; D003605:D-Amino-Acid Oxidase