Luciferin-luciferase reaction kinetics

Luminescence reaction (Viviani et al., 2002a) The luciferin-luciferase luminescence reaction was carried out in 0.1 M Tris-HCl, pH 8.0, containing 2mM ATP and 4mM Mg2+. Mixing luciferase with luciferin and ATP resulted in an emission of light with rapid onset and a kinetically complex decay. Further additions of fresh luciferase, after the luminescence has decayed to about 10% of its maximum value, resulted in additional luminescence responses similar to the initial one (Fig. 1.15). According to the authors, the repetitive light emission occurred in consequence of the inhibition of luciferase by a reaction product, as seen in the case of the firefly system (McElroy et al., 1953). The luminescence spectrum showed a peak at 487nm (Fig. 1.16). 

The main conclusion from this study is that both PP and LM firefly luciferases have similar kinetic mechanisms characterized by significant inactivation of the enzyme induced by its interaction with the substrates. The difference in kinetic properties for both enzymes is mainly in the reaction rates for formation and dissociation of the luciferin-luciferase comlex. The addition of pyrophosphate to the reaction mixture increases the reaction yield due to enhanced regeneration of active enzyme from the enzyme-product complex. 

Some years ago I demonstrated that such a biochemical flash can be produced in the test tube.9,15 If oxygen is excluded from a firefly luciferase reaction mixture and then added rapidly back, a bright flash occurs, some 100 to 200 times brighter than the baseline intensity (Fig. 3). This comes from the reaction of the luciferyl adenylate active intermediate accumulated in the absence of oxygen. Note that the decay of the flash is not due to the removal of oxygen, but to the utilization of the luciferase-peroxide intermediate, so the baseline returns to a low level (Fig. 4), defined by the slow rate of reaction of ATP with luciferin. It is well known that the kinetics of firefly flashes are species specific and of functional importance in courtship communication, fixed by the rate constant for the first order decay of the peroxide intermediate formed from the adenylate. 

Fig. 7.3.4 Kinetic profiles of the Diplocardia bioluminescence reaction, when Diplocardia luciferase, H2O2, or Diplocardia luciferin was injected last. In each case, 0.1 ml of the last component was injected into 0.9 ml of the mixture of other components, to give the final concentrations Diplocardia luciferase, 0.1 unit/ml Diplocardia luciferin, 32 mM and H2O2, 32 mM, in 0.1 M potassium phosphate buffer, pH 7.5. From Rudie et al., 1981, with permission from the American Chemical Society.

The light yield and kinetics of in vitro luminescence vary with the order of the addition of components, as shown in Fig. 7.3.4 (Rudie et al., 1981). The light yield and initial rate are highest when luciferin and peroxide have been preincubated and the reaction is initiated by the injection of luciferase. Based on this and some other data, it was concluded that the true substrate of the Diplocardia luciferase is the H2O2 adduct of luciferin shown below (Rudie et al., 1981). 

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