Journal
FEBS JOURNAL
Volume 272, Issue 20, Pages 5206-5216Publisher
WILEY
DOI: 10.1111/j.1742-4658.2005.04895.x
Keywords
coenzyme A; dehydroluciferyl-adenylate; dehydroluciferyl-coenzyme A; dephosphocoenzyme A; firefly luciferase
Categories
Ask authors/readers for more resources
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 mu m). 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available