194 



BIOCHEMISTRY OF FIREFLY LUMINESCENCE 



inactive complex or complexes and the mode of the splitting by pyro- 

 phosphate. The simplest inactive complex would presumably be 

 formed of luciferin-luciferase-Mg-ATP-Mg-pyrophosphatase. Pyrophos- 

 phate could split such a complex to give rise to both active and in- 

 active intermediates containing luciferase. The initial light intensity 

 obtained with the addition of pyrophosphate would then be a meas- 

 ure of the active intermediate formed. The secondary peak of lumin- 

 escence, after the addition of pyrophosphate, would represent the slow 

 release of luciferase from an inhibitory complex with pyrophosphate. 

 The schematic relationships are shown in Fig. 25. 



LHg+E + Mg *• ATP 



1 1 



LHg-E'Mg-ATP 



(ACTIVE INTERMEDIATE) » + 0, » LIGHT 



Mg 

 Pr 



P; +Mg-Pr 



t 

 LHj-E-Mg-ATP + PQP-Mg-Pr 



LH,-E-Mg-ATP'Mg-Pr -f p-q-p °' 



(INACTIVE COMPLEX) « L Hj- E' Mg- POP + ATP- Mg- Pr 



[INHieiTORvl 

 COMPLEX 



CUNIKUL -■ 



MECHANISM 



ACETYLCHOLINE 



CHOLINE + AC 



♦ CoA 



♦ POP + Ac'Co A + Ad. 



Fig. 25. Scheme for the coniplexing reaction and the function of pyrophosphatase 

 and pyrophosphate in firefly luminescence. 



The effect of various pyrophosphatase inhibitors, such as Mn, Ca, 

 and F, on the luminescent response to pyrophosphate can be ex- 

 plained on such a hypothesis. The quantitative relationships between 

 pyrophosphate, pyrophosphatase, luciferase, and Mg++ presented 

 support this interpretation. The results also indicate that triphosphate 

 has an action similar to that proposed for pyrophosphate. However, 

 since triphosphate is not hydrolyzed by the enzyme preparations, the 

 steady-state luminescence remains higher after the addition of this 

 compound. The results suggest that the equilibrium between active 

 intermediate and inactive complex is shifted in favor of the former 

 when triphosphate is added. Since additional pyrophosphatase can 



