Pyrophosphate and tripolyphosphate affect firefly luciferase luminescence because they act as substrates and not as allosteric effectors.
The activating and stabilizing effects of inorganic pyrophosphate, tripolyphosphate and nucleoside triphosphates on firefly luciferase bioluminescence were studied. The results obtained show that those effects are a consequence of the luciferase-catalyzed splitting of dehydroluciferyl-adenylate, a powerful inhibitor formed as a side product in the course of the bioluminescence reaction. Inorganic pyrophosphate, tripolyphosphate, CTP and UTP antagonize the inhibitory effect of dehydroluciferyl-adenylate because they react with it giving rise to products that are, at least, less powerful inhibitors. Moreover, we demonstrate that the antagonizing effects depended on the rate of the splitting reactions being higher in the cases of inorganic pyrophosphate and tripolyphosphate and lower in the cases of CTP and UTP. In the case of inorganic pyrophosphate, the correlation between the rate of dehydroluciferyl-adenylate pyrophosphorolysis and the activating effect on bioluminescence only occurs for low concentrations because inorganic pyrophosphate is, simultaneously, an inhibitor of the bioluminescence reaction. Our results demonstrate that previous reports concerning the activating effects of several nucleotides (including some that do not react with dehydroluciferyl-adenylate) on bioluminescence were caused by the presence of inorganic pyrophosphate contamination in the preparations used.
Fontes
Rui
R
 Serviço de Bioquímica (U38-FCT), Faculdade de Medicina da Universidade do Porto, Portugal.
Fernandes
Diogo
D
 Serviço de Bioquímica (U38-FCT), Faculdade de Medicina da Universidade do Porto, Portugal.| Departamento de Química, Faculdade de Ciências da Universidade do Porto, Centro de Investigação em Química (UP), Portugal.
Peralta
Filipe
F
 Departamento de Química, Faculdade de Ciências da Universidade do Porto, Centro de Investigação em Química (UP), Portugal.
Fraga
Hugo
H
 Departamento de Química, Faculdade de Ciências da Universidade do Porto, Centro de Investigação em Química (UP), Portugal.
Maio
Inês
I
 Serviço de Bioquímica (U38-FCT), Faculdade de Medicina da Universidade do Porto, Portugal.
Esteves da Silva
Joaquim C G
JCG
 Departamento de Química, Faculdade de Ciências da Universidade do Porto, Centro de Investigação em Química (UP), Portugal.
D000494:Allosteric Regulation / Q000502:physiology; D000818:Animals; D011756:Diphosphates / Q000737:chemistry* / Q000378:metabolism; D048888:Fireflies / Q000201:enzymology*; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000378:metabolism; D049449:Luminescence; D049408:Luminescent Agents / Q000737:chemistry* / Q000378:metabolism; D011122:Polyphosphates / Q000737:chemistry* / Q000378:metabolism; D011755:Pyrophosphatases / Q000737:chemistry / Q000502:physiology; D013379:Substrate Specificity / Q000502:physiology
D011756:Diphosphates; D049408:Luminescent Agents; D011122:Polyphosphates; D049409:Luciferases, Firefly; D011755:Pyrophosphatases; C005692:triphosphoric acid