11. 1). Mcelroy and h. h. seijc.er 231 



AtlclitioiKil cvidciuc that only one active site is involved in light 

 prothiction has Ijeen obtained by studying tlic flasli lieight in the 

 presence ol LH^-AMP and LHj and ATP with large variations in tlie 

 enzyme concentration. The results ol such an cxj)erinicnt are pre- 

 sented in Fig. \). 11 more than one lucilerin or LH.j-AMP were re- 

 (|uircd lor light emission one might expect an inhibition of the flash 

 height with excess enzyme. However, the evidence indicates that a 

 l.OOO-fold excess of enzyme concentration relative to the substrate 

 concentration has no effect on the flash Iieight. We conclude from 

 such evidence that there is one active LH^-AAfP formed on tlie enzyme 

 surface, and that this interacts \\[\\\ oxygen in some way to j)roduce 

 the excited state. 



The Reaction of Coenzyme A with Enzyme-L-AMP Complex 



When coenzyme A is added to a reaction mixture in which [LHo] 

 > [E] and which has been allowed to proceed to the region of slow- 

 light decay, there is an immediate rise in the light intensity, a rise 

 directly proportional to the coenzyme A concentration. After the 

 initial rise due to the coenzyme A addition the luminescence will 

 continue at this high level of light emission and finally return to 

 the normal slow decay level. The duration of this stimidated light 

 emission is also directly proportional to the initial CoA concentra- 

 tion. If CoA is added initially to the reaction mixture there is 

 no effect on the initial light intensity, but the reaction mixture con- 

 taining CoA always remains at a higher value than an identical mix- 

 ture without CoA (1) . 



CoA is very specific in its stimulatory effect. Neither dephospho- 

 CoA, nor phosphopantothine, nor cysteine is effective in this respect. 

 Tlie inhibitory effect of oxyluciferin or oxyluciferyl-adenylate can be 

 removed by the addition of CoA. A considerable amount of evidence 

 now indicates that CoA stimulates light production by reacting with 

 the E-L-AMP to produce AMP, oxyluciferyl-CoA, and free enzyme. 

 The results diagrammed in Fig. 10 indicate that L-CoA can be pro- 

 duced from oxyluciferin if ATP, luciferase, and magnesium ion are 

 present in the reaction mixture. Spectrophotofluorometry provides 

 a good means of measining tlie production of L-CoA since its fluores- 

 cence intensity is approximately 2 per cent of that of oxyluciferin. 

 The reaction producing L-CoA has i)een shown to be enzymatically 

 reversible by obtaining a net synthesis of ATP from L-CoA, AMP, 

 and pyrophosphate; by measuring a coenzyme-A-dependent incorpora- 

 tion of C^^-labeled AMP into ATP; and by measuring the formation 



