A study on apoenzyme from Rhodotorula gracilis D‐amino acid oxidase

Abstract

The apoenzyme of d ‐amino acid oxidase from Rhodotorula gracilis was obtained at pH 7.5 by dialyzing the holoenzyme against 2 M KBr in 0.25 M potassium phosphate, 0.3 mM EDTA, 5 mM 2‐mercaptoethanol and 20% glycerol. To recover a reconstitutable and highly stable apoprotein, it is essential that phosphate ions and glycerol be present at high concentrations. Apo‐ d ‐amino acid oxidase is entirely present as a monomeric protein, while the reconstituted holoenzyme is a dimer of 79 kDa. The equilibrium binding of FAD to apoprotein was measured from the quenching of flavin fluorescence and by differential spectroscopy: a K d of 2.0 × 10 −8 M was calculated. The kinetics of formation of the apoprotein‐FAD complex were studied by the quenching of protein and flavin fluorescence, by differential spectroscopy and by activity measurements. In all cases a two‐stage process was shown to be present with a fairly rapid first phase, followed by a slow secondary change which represents only 4–6% of the total recombination process. In no conditions was a lag in the recovery of maximum catalytic activity observed. The process of FAD binding to yeast d ‐amino acid oxidase appears to be of the type Apo + FAD ⇋ 2 holoenzyme, even though the existence of a transient intermediate not detectable under our conditions cannot be ruled out.