RELATIONSHIP BETWEEN MOLECULAR CONFORMATION AND BINDING OF FLAVIN-ADENINE DINUCLEOTIDE IN GLUCOSE OXIDASE

1. 1.|Evidence has been obtained from biophysical and kinetic studies that flavin-adenine dinucleotide (FAD), the prosthetic group of glucose oxidase, stabilizes the three-dimensional structure of the enzyme. 2. 2.|FAD is bound tightly to the enzyme and cannot be removed by dialysis at neutral pH. Protein free from flavin was prepared by treatment with acidified (NH4)2SO4. 3. 3.|The binding of the prosthetic group to the apoenzyme has been followed kinetically in terms of the reappearance of oxidase activity and by the quenching of FAD fluorescence and the enhancement of FAD absorbance. The primary bimolecular step was investigated by studies of the polarization of FAD fluorescence. Binding also involved at least one and probably two unimolecular steps associated with changes in protein conformation. 4. 4.|The apoenzyme has the same molecular weight as the holoenzyme but a different sedimentation coefficient, intrinsic viscosity and sensitivity to proteolysis. 5. 5.|It is proposed that in the absence of FAD, the protein exists mainly with the molecular conformation of a loose flexible coil. When FAD is added, the protein is converted to a compact nearly spherical form possessing enzyme activity. © 1969.