224 LIGHT AND LIIE 



oxyluciferyl-adenylic acid complex to give free enzyme, ATP, and 

 free oxyluciferin. The free enzyme in the presence of ATP is now- 

 available to catalyze the oxidation of a second luciferin molecide. 

 We believe that the stimnlation by pyrophosphate in the manner 

 discussed above is the mechanism of control of the firefly flash (9, 15). 

 We have discussed previously the existence of such inactive com- 

 plexes within enzyme-catalyzed reactions and have considered their 

 importance as controlling mechanisms not only in the firefly flash 

 but in other enzyme-catalyzed processes (12) . 



Response to LH,-AMP 



Probably the best evidence for the proposed scheme in Fig. 1 is 

 the demonstration that LHo-AMP, formed chemically by the con- 

 densation of LH^ and AMP, can act as an active substrate in the 

 presence of the enzyme and without ATP or magnesiinii ion to pro- 

 duce light. Starting with LHo-AMP and crystalline luciferase in 

 solution, the only other requirement for light emission is oxygen. 

 Free oxyluciferin has no inhibitory effect on the rate of light emission. 

 However, ATP gives a slight inhibition of the rate of light emission 

 which becomes much more pronounced in the presence of magnesiimi 

 ion. 



The product of the enzymatic light reaction initiated with LHg- 

 AMP has been identified as the oxyluciferyl-adenylate derivative. 

 The inhibitory effect of oxyluciferyl-adenylate on the flash height 

 is even more pronounced in the absence of ATP and free LH2 when 

 starting with LH^-AMP as the reactant. This is shown in Fig. 4, 

 where light production from LHo-AMP is compared to that initiated 

 with ATP and an equal quantity of luciferin. The continuous pro- 

 duction of light from ATP and luciferin suggests that either ATP or 

 luciferin, or both, are capable of removing the LAMP inhibitor 

 from the enzyme surface. This is not the case with LHo-AMP. The 

 fact that the secondary addition of luciferase to the reaction mixture 

 showed a burst of light indicates that excess LHo-AMP was present. 

 The production of jjyrophosphate in the presence of ATP and LHo 

 because of the initial activating reaction is undoubtedly important in 

 sustaining a low but continuous light emission. As the results in 

 Fig. 5 indicate, the addition of pyrophosphate along with LHo-AMP 

 to the luciferase solution decreases the initial rate. However, the total 

 light production has been greatly increased due to the release of 

 enzyme from the E-L-AMP inhibited complex. It should be noted 

 that if both magnesiinii ions and pyrophosphate are added, there is 



