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J. Biochem, 1990, Vol. 107, No. 5 726-731
© 1990 Japanese Biochemical Society

research-article

Proton Release from Flavoprotein D-Amino Acid Oxidase on Complexation with the Zwitterionic Ligand, Trigonelline1

Yasuzo Nishina, Kyosuke Sato and Kiyoshi Shiga

Department of Physiology, Kumamoto University Medical School Honjo, Kumamoto, Kumamoto 860

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.

1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.


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