Apoenzyme from Cryptococcus humicolus UJ1 D-aspartate oxidase

The apoenzyme of D-aspartate oxidase from Cryptococcus humicolus UJ1 was ob tained by dialyzing the holoenzyme against 3 M KBr in 250 mM potassium phos phate (pH 7.0), 0.3 mM EDTA and 5 mM 2-mercaptoethanol, followed by gel fil tration on Superdex 200 to separate from the remaining holoenzyme. Apo-D-as partate oxidase is entirely present as a monomeric protein of 40 kDa, while the reconstituted holoenzyme is a tetramer of 160 kDa. The equilibrium bin ding of FAD to apoprotein was measured from the quenching of flavin fluores cence: a very small value of K-d (8.2 x 10(-12) M) was calculated. The kine tics of formation of the apoprotein-FAD complex was studied by the quenchin g of protein and flavin fluorescence and by activity measurements. The reac tion apparently proceeded in two stages, a rapid first phase, followed by a slower secondary phase. The rapid phase was observed by the change in prot ein 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 que nching and the appearance of catalytic activity. (C) 2000 Elsevier Science B.V. All rights reserved.