A study on apoenzyme from Rhodotorula gracilis D-amino acid oxidase.
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 Kd of 2.0 x 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 in equilibrium holoenzyme, even though the existence of a transient intermediate not detectable under our conditions cannot be ruled out.
Department of General Physiology and Biochemistry, University of Milano, Italy.
P
P
Casalin
L
L
Pollegioni
B
B
Curti
M
M
Pilone Simonetta
D001665:Binding Sites; D003605:D-Amino-Acid Oxidase / Q000737:chemistry*; D005453:Fluorescence; D007527:Isoenzymes / Q000737:chemistry*; D012248:Rhodotorula / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000737:chemistry
D007527:Isoenzymes; D013438:Sulfhydryl Compounds; D003605:D-Amino-Acid Oxidase