Coenzyme A affects firefly luciferase luminescence because it acts as a substrate and not as an allosteric effector

• Type: Article in International Scientific Journal
• Date: 2005

Authors

Fraga, H (Author) Other; Fernandes, D (Author) Other; Fontes, R (Author) FMUP; da Silva, JCGE (Author) FCUP

Journal

Title: FEBS Journal

Vol. 272

Pages: 5206-5216

ISSN: 1742-464X

Publisher: Wiley-Blackwell

Indexing

ISI Web of Knowledge - 65 Citations

Scopus - 68 Citations

Scientific classification

FOS: Natural sciences > Biological sciences

Other information

Authenticus ID: P-000-12E

DOI: 10.1111/j.1742-4658.2005.04895.x

Abstract (EN): 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.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 11