19F-NMR study on the interaction of fluorobenzoate with porcine kidney D-amino acid oxidase.

The interactions of competitive inhibitors, o-, m-, and p-fluorobenzoates, with porcine kidney D-amino acid oxidase (DAO) were studied by 19F-NMR spectroscopy. The 19F-signals of DAO-bound fluorobenzoates were observed as considerably broadened peaks. The chemical shifts, which are referenced to 20 mM NaF in 50 mM sodium phosphate, pH 7.0, were 6.0, 8.2, and 11.9 ppm for free o-, m-, and p-fluorobenzoates, respectively, while those of o-, m-, and p-fluorobenzoates bound to DAO were 12.5, 5.4, and 13.1 ppm, respectively. The 19F-signals of bound o- and p-fluorobenzoates were downfield-shifted relative to those of the free species, whereas the 19F-resonance of m-fluorobenzoate was up-field shifted from that of the free ligand. The magnitude of the chemical shift difference between the free and bound forms decreases in the order of o-, m-, and p-fluorobenzoates. The remarkably large downfield shift of the o-fluorobenzoate when bound to DAO was attributed to the close proximity of the ortho-fluorine atom to the flavin nucleus in comparison with meta- or para-fluorine. The pH-dependences of the 19F-resonances of o-, m-, and p-fluorobenzoates were observed and the pKa values of 3.33, 3.80, and 4.05 were obtained for the carboxyl groups of o-, m-, and p-fluorobenzoates, respectively. It was observed that the 19F-resonances of o- and p-fluorobenzoates are highly sensitive to the ionic state of the carboxyl group, while that of m-fluorobenzoate was moderately sensitive.