Apoenzyme from Cryptococcus humicolus UJ1 d-aspartate oxidase

The apoenzyme of d-aspartate oxidase from Cryptococcus humicolus UJ1 was obtained by dialyzing the holoenzyme against 3 M KBr in 250 mM potassium phosphate (pH 7.0), 0.3 mM EDTA and 5 mM 2-mercaptoethanol, followed by gel filtration on Superdex 200 to separate from the remaining holoenzyme. Apo-d-aspartate oxidase is entirely present as a monomeric protein of 40 kDa, while the reconstituted holoenzyme is a tetramer of 160 kDa. The equilibrium binding of FAD to apoprotein was measured from the quenching of flavin fluorescence: a very small value of Kd (8.2×10−12 M) was calculated. The kinetics of formation of the apoprotein–FAD complex was studied by the quenching of protein and flavin fluorescence and by activity measurements. The reaction apparently proceeded in two stages, a rapid first phase, followed by a slower secondary phase. The rapid phase was observed by the change in protein fluorescence and an initial rapid phase of decrease in FAD fluorescence, representing at least initial binding of FAD to the monomeric apoprotein. The slower phase correlated with a secondary phase of FAD fluorescence quenching and the appearance of catalytic activity.