¹³C-NMR Studies of Porcine Kidney D-Amino Acid Oxidase Reconstituted with ¹³C-Enriched Flavin Adenine Dinucleotide. Effects of Competitive Inhibitors

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D-Amino acid oxidase (DAO) from porcine kidney was reconstituted with FAD's which were enriched with ¹³C at the 2-, 4-, 4a-, and lOa-positions of the isoalloxazine moiety, and ¹³C-NMR spectra of the reconstituted DAO were measured in the absence and presence of various competitive inhibitors. When compared to the corresponding chemical shifts of FMN at infinite dilution (Moonen, C. T. W. et al. (1984) Biochemistry 23, 4859-4867), the resonances of C (2), C (4), C (4a), and C (1Oa) of FAD bound to apoDAO were all shifted to higher field. However, when the reconstituted DAO was complexed with o-, m-, p-aminobenzoate, or benzoate, each of the four ¹³C-signals underwent a different change in chemical shift. In these changes we observed no characteristics which distinguish DAO-aminobenzoate complexes from DAO-benzoate complex, even though o-, m-, and p-aminobenzoates are known to form charge-transfer complexes with DAO. The signals due to 2- and 4-¹³C were more sensitive to the formation of the inhibitor-DAO complexes than those of the other carbon atoms. These findings suggest the modulation of the hydrogen bonds at the oxygen atoms of C(2)=O and C(4)=O with the protein moiety as the result of the inhibitor-binding.