Influence of FAD structure on its binding and activity with the C406A mutant of recombinant human liver monoamine oxidase A

The FAD binding site of human liver monoamine oxidase A (MAO A) has been in vestigated by mutagenesis of the amino acid site of covalent FAD attachment (Cys-406) to an alanyl residue. Expression of the C406A mutant in Saccharo myces ces cerevisiae results in the formation of an active enzyme, as found previously with the rat liver enzyme. The activity of this mutant enzyme i s labile to solubilization, thus requiring all experiments to be done with membrane preparations. C406A MAO A was expressed in a rib 5(-) strain of S. cerevisiae in the presence of 16 different riboflavin analogues. Inactive apoC406A MAO A is formed by induction of the enzyme in the absence of ribof lavin. FAD but not FMN or riboflavin restores catalytic activity with an ap parent K-d of 62 +/- 5 nM. The results from both in vivo and in vitro recon stitution experiments show increased activity levels (up to similar to 7-fo ld higher) with those analogues exhibiting higher oxidation-reduction poten tials than normal flavin and decreased activity levels with analogues exhib iting lower potentials. Analogues with substituents on the pyrimidine ring bind to C406A MAO A more weakly than normal FAD, suggesting specific intera ctions with the N(3) and N(1) positions. Analogues with substituents in the 7 and 8 positions bind to C406A MAO A with affinities comparable with that of normal FAD. These results are discussed in regard to functional signifi cance of 8 alpha-covalent binding of flavins to proteins.