title: Fluorescence properties of the copper enzyme galactose oxidase and its tryptophan-modified derivatives.
abstract: Galactose oxidase contains a single nonblue Cu(II) atom and 18 tryptophan residues per molecule. Removal of the copper atom reveals that it has an approximately 29% quenching effect on the relative quantum yield of fluorescence. While saturating concentrations of the sugar substrate of galactose oxidase also reduce the quantum yield, the second substrate, oxygen, has no significant effect on fluorescence in the absence of the sugar substrate. N-Bromosuccinimide (NBS) inactivates galactose oxidase as two tryptophans are oxidized (Kosman, D. J., Ettinger, M. J., Bereman, R. D., and Giordano, R. S. (1977), Biochemistry, 16). Oxidation of two tryptophans also leads to a disproportionately large decrease in fluorescence intensity. A 23% reduction in quantum yield with blue-shift occurs with oxidation of 0.85 tryptophan equiv and a further 25% quenching is obtained as the reaction proceeds to 2.0 residues oxidized. Fluorescence experiments with the modified enzyme show that it contains at least one tryptophan residue which is unreactive towards NBS, but which also interacts with the Cu(II) atom and substrate. These results taken together substantiate the postulate that one or more tryptophan residues, the Cu(II) atom, and the sugar substrate mutually interact within the native enzyme. Energy-transfer calculations suggest that this residue(s) which must be within a relatively hydrophobic environment is at least 12 A from the Cu(II) atom.
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism; D001665:Binding Sites; D001974:Bromosuccinimide; D003300:Copper / Q000032:analysis; D005692:Galactose Oxidase / Q000378:metabolism; D008433:Mathematics; D003904:Mitosporic Fungi / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
chemical_list: D003300:Copper; D014364:Tryptophan; D000429:Alcohol Oxidoreductases; D005692:Galactose Oxidase; D001974:Bromosuccinimide