The reaction process of firefly bioluminescence was studied by photolyzing caged-ATP to adenosine triphosphate (ATP) within 100 ms. The intensity of luminescence increases markedly to reach a maximum within 1 s, maintains almost the same intensity up to 5 s and then decays monotonically. The rise γ1 and decay γ2 rate constants were determined to be about 5 s−1 and 1 × 10−2 s−1, respectively, so as to phenomenologically fit the time course. A second luminescence peak appears after around 350 s. The dependence of the rate constants on the concentrations of reactants and a viscous reagent revealed that two kinds of reaction contribute the observed time course: (1) an intrinsic reaction by ATP photolyzed from caged-ATP that is already trapped in luciferase; and (2) a diffusion-controlled reaction by free ATP in the buffer solution outside luciferase. Numerical analysis based on reaction kinetics related γ1 and γ2 to the rate constants of a three-step reaction model, and accurately described the effects of concentration of reactants and a viscous reagent on the time courses of bioluminescence. Thus, it has been clearly concluded that the binding mode of caged-ATP at the catalytic center of luciferase is very different from that of ATP.
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