Differential effects of 8-anilino-1-naphthalenesulfonate upon binding of oxidized and reduced flavines by bacterial luciferase.
Upon binding to bacterial luciferase, both the absorption and the fluorescence excitatiom maxima of 8-anilino-1-naphthalensulfonate (ANS) shift from 353 to 370 nm while the fluorescence emission optimum shifts from 540 to 480 nm, and the fluorescence quantum yield increases from 0.003 to 0.39, indicating that the environment of the ANS binding site is hydrophobic. ANS binds to luciferase with dissociation constants of 1.9 X 10(-5) and 2.3 X 10(-5) M at 5 and 23 degrees, repsectively. As with both oxidized flavine mononucleotide (FMN) and reduced flavine mononucleotide (FMNH2), ANS also binds to luciferase with a stoichiometry of 1 site per dimeric luciferase molecule. ANS acts as a luciferase inhibitor, competitive with FMNH2, with an inhibitor constant of 2.3 X 10(-5) M at 23 degrees. However, the binding of ANS does not significantly displace FMN from binding to luciferase. Interactions of FMN and FMNH2 with luciferase are thus differentially regulated by the ANS binding.
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology*; D001665:Binding Sites; D001667:Binding, Competitive; D005486:Flavin Mononucleotide / Q000378:metabolism*; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D046911:Macromolecular Substances; D005486:Flavin Mononucleotide; D008156:Luciferases