Reconstitution of flavin-adenine dinucleotide in the apoenzyme of glucose oxidase.

The reconstitution reaction of glucose oxidase from FAD and apoenzyme was studied : (1) Unimolecular reaction takes part in the reaction of glucose apooxidase with FAD as a rate limiting step. (2) At the very initial stage of the reaction, intensity of the fluorescence derived from FAD was observed to increase rapidly and to be quenched slowly after reaching the maximum state. (3) When glucose apooxidase was incubated with several nucleosides, nucleotides, FMN and riboflavin, FAD incorporation into apoenzyme was strongly inhibited by adenosine, moderately by guanosine and adenine, and was not inhibited by FMN and riboflavin. (4) An allosteric transition induced by FAD molecule itself probably occurred in the course of the reconstitution reaction, and the second FAD molecule seemed to be likely incorporated spontaneously soon after the first FAD was incorporated. (5) The mechanism that FAD is first bound to the apoprotein via adenosine residue was confirmed judging from the considerable strength of adenosine and the relative weakness of FMN or riboflavin for the inhibitory effect of the reconstitution reaction.