Coenzyme A affects firefly luciferase luminescence because it acts as a substrate and not as an allosteric effector.
The effect of CoA on the characteristic light decay of the firefly luciferase catalysed bioluminescence reaction was studied. At least part of the light decay is due to the luciferase catalysed formation of dehydroluciferyl-adenylate (L-AMP), a by-product that results from oxidation of luciferyl-adenylate (LH2-AMP), and is a powerful inhibitor of the bioluminescence reaction (IC50 = 6 nm). We have shown that the CoA induced stabilization of light emission does not result from an allosteric effect but is due to the thiolytic reaction between CoA and L-AMP, which gives rise to dehydroluciferyl-CoA (L-CoA), a much less powerful inhibitor (IC50 = 5 microm). Moreover, the V(max) for L-CoA formation was determined as 160 min(-1), which is one order of magnitude higher than the V(max) of the bioluminescence reaction. Results obtained with CoA analogues also support the thiolytic reaction mechanism: CoA analogues without the thiol group (dethio-CoA and acetyl-CoA) do not react with L-AMP and do not antagonize its inhibitor effect; CoA and dephospho-CoA have free thiol groups, both react with L-AMP and both antagonize its effect. In the case of dephospho-CoA, it was shown that it reacts with L-AMP forming dehydroluciferyl-dephospho-CoA. Its slower reactivity towards L-AMP explains its lower potency as antagonist of the inhibitory effect of L-AMP on the light reaction. Moreover, our results support the conjecture that, in the bioluminescence reaction, the fraction of LH2-AMP that is oxidized into L-AMP, relative to other inhibitory products or intermediates, increases when the concentrations of the substrates ATP and luciferin increases.
Fraga
Hugo
H
Departmento de Química, Faculdade de Ciências da Universidade do Porto, Portugal.
Fernandes
Diogo
D
Fontes
Rui
R
Esteves da Silva
Joaquim C G
JC
D000105:Acetyl Coenzyme A / Q000737:chemistry; D000214:Acyl Coenzyme A / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000494:Allosteric Regulation; D000818:Animals; D003065:Coenzyme A / Q000737:chemistry*; D004791:Enzyme Inhibitors; D049454:Firefly Luciferin / Q000737:chemistry; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry*; D049449:Luminescence
D000214:Acyl Coenzyme A; D004791:Enzyme Inhibitors; C107793:dehydroluciferin; C501929:luciferyl-coenzyme A; C015787:dephosphocoenzyme A; D049454:Firefly Luciferin; D000105:Acetyl Coenzyme A; D000255:Adenosine Triphosphate; D049409:Luciferases, Firefly; D003065:Coenzyme A