| dc.description.abstract | Firefly luciferase catalyzes a bioluminescent oxidation of luciferin. During the first step of the reaction, when luciferin interacts with ATP, a molecule of luciferyl adenylate is formed and a molecule of pyrophosphate is produced. The luciferyl adenylate is further oxidized by molecular oxygen with the intermediate formation of a cyclic peroxide, dioxetanone, and a molecule of AMP. The dioxetanone is decarboxylated as a result of intramolecular conversions to produce an electron-excited molecule of oxyluciferin which is accompanied with emission of a quantum of visible light (λₘₐₓ= 562-570 nm). The experiments reported here show that luciferase binds to luciferin with an apparent equilibrium dissociation constant, of 24.7 μM at 25°C in 100 mM Tris-acetate buffer, pH 7.8, 2 mM EDTA, and 10 mM MgSO₄. Analysis of the binding at temperatures between 3° and 25°C indicates an enthalpy of binding (ΔHₐ) of -11.5 kcal/mol. Although a solution of luciferin is highly fluorescent, the binding of luciferin to luciferase does not significantly change the fluorescence of the luciferin. However, the enzyme's comparatively weak fluorescence signal does change significantly on the binding of luciferin. This spectral property of the enzyme allows a sensitive method for the determination of the equilibrium dissociation constant of luciferin and luciferase. | en |
