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 µm). Moreover, the Vmax for L‐CoA formation was determined as 160 min−1, which is one order of magnitude higher than the Vmax 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.

Firefly luciferase catalyzes the synthesis of H2O2 from the same substrates as the bioluminescence reaction: ATP and luciferin (D-LH2). About 80% of the enzyme-bound intermediate D-luciferyl adenylate (D-LH2-AMP) is oxidized into oxyluciferin, and a photon is emitted during this reaction. The enzyme pathway responsible for the generation of H2O2 is a side reaction in which D-LH2-AMP is oxidized into dehydroluciferyl adenylate (L-AMP). Like the bioluminescence reaction, the luciferase-catalyzed synthesis of H2O2 and L-AMP is a stereospecific process, involving only the natural D enantiomer. However, the intramolecular electron transfer postulated as essential to the light emission process is not involved in this side reaction.

Firefly luciferase (LUC, EC 1.13.12.7) is an enzyme that catalyses the oxidation of firefly luciferin (LH 2 ) giving rise to light [1][2][3][4]. The bioluminescence reaction involves the formation, from LH 2 and ATP, of an enzyme-bound adenylyl intermediate (LUCAELH 2 -AMP) and its subsequent oxidation with the release of AMP, CO 2 and oxyluciferin in an electronically excited state.In addition to oxyluciferin, dehydroluciferyl-adenylate (L-AMP) is also a LH 2 -AMP oxidation product in a side reaction. Its formation from the LUCAELH 2 -AMP 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.Abbreviations Ap 4 C, adenosine(5¢)tetraphospho(5¢)cytidine; Ap 4 N, adenosine(5¢)tetraphospho(5¢)nucleoside; Ap 4 U, adenosine(5¢)tetraphospho(5¢)uridine; DE, degree of effect; IP, ion-pair; L, dehydroluciferin; L-AMP, dehydroluciferyl-adenylate; L-CoA, dehydroluciferyl-CoA; LH 2, firefly luciferin; LH 2 -AMP, luciferyl-adenylate; LUC, firefly luciferase; NTP, nucleoside triphosphate; P3, tripolyphosphate; p4A, adenosine tetraphosphate; PPase, inorganic pyrophosphatase; PPi, inorganic pyrophosphate; RLU, relative light units.

This work introduces a novel resource allocation technique for dealing with linear and periodically time‐varying power line channels when an orthogonal frequency division multiplexing scheme is applied. By exploiting the correlations within one cycle of the mains signal, among cycles of mains signal and a combination of these connections, the proposed technique can offer three distinct trade‐offs between computational complexity reduction and data‐rate loss. Numerical results, which are based on measured data, are used to analyse these trade‐offs, when these inter‐cycle and intra‐cycle relationship are taken into account. Also, we verify that the use of the normalised signal‐to‐noise ratio incurs very low performance degradation, and because of computational complexity rationale, its use is strongly recommended. Additionally, we show that those cases, in which the correlations among the cycles of the mains signal is relevant, offer the best trade‐off between computational complexity reduction and data‐rate loss. Finally, we show that the proposed technique can achieve the optimal data‐rate and offers substantial improvements in terms of computational complexity when compared with existing approaches, including the Institute of Electrical and Electronics Engineers 1901 standard. Copyright © 2015 John Wiley & Sons, Ltd.

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