Rui Fontes

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 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 Produces Hydrogen Peroxide as a Coproduct in Dehydroluciferyl Adenylate Formation

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.

Dehydroluciferyl-AMP is the main intermediate in the luciferin dependent synthesis of Ap4A catalyzed by firefly luciferase.

It was previously assumed that E·LH2‐AMP was the intermediate complex in the synthesis of Ap4A catalyzed by firefly luciferase (EC 1.13.12.7), when luciferin (LH2) was used as cofactor. However, here we show that LH2 is partly transformed, shortly after the onset of the luciferase reaction, to dehydroluciferin (L) with formation of an E·L‐AMP complex which is the main intermediate for the synthesis of Ap4A. Formation of three more derivatives of LH2 were also observed, related to the production of light by the enzyme. CoA, a known stimulator of light production, inhibits the synthesis of Ap4A by reacting with the E·L‐AMP complex and yielding L‐CoA.

Identification of enzyme produced firefly oxyluciferin by reverse phase HPLC

Firefly oxyluciferin (2-(6′-hydroxybenzothiazolyl)-4-hydroxythiazole) was chemically synthesized and characterized by means of 13C and 1H NMR, UV–vis spectrometry and RP-HPLC using different pH elution conditions. One of the chromatographic peaks observed in luciferase-catalyzed reaction mixtures was identified as corresponding to oxyluciferin.

Identification of Luciferyl Adenylate and Luciferyl Coenzyme A Synthesized by Firefly Luciferase

The firefly luciferase reaction intermediate luciferyl adenylate was detected by RP‐HPLC analysis when the luciferase reaction was performed under a nitrogen atmosphere. Although this compound is always specified as an intermediate in the light‐production reaction, this is the first report of its identification by HPLC in a luciferase assay medium. Under a low‐oxygen atmosphere, luciferase can catalyze the synthesis of luciferyl coenzyme A from luciferin, ATP, and coenzyme A, but in air dehydroluciferyl coenzyme A was produced. The luciferase‐catalyzed synthesis of these coenzyme A derivatives may be a consequence of the postulated recent evolutionary origin of firefly luciferases from an ancestral acyl‐coenzyme A synthetase.

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.

pH opposite effects on synthesis of dinucleoside polyphosphates and on oxidation reactions catalyzed by firefly luciferase

Previous results have shown that an oxidizing product of firefly luciferin, dehydroluciferyl‐adenylate, is the main intermediate in the process of synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase (EC 1.13.12.7). However, we have found that the pH effects on the luciferase oxidizing processes and on the synthesis of dinucleoside polyphosphate are opposite: acidic assay media enhance the synthesis of dinucleoside polyphosphate and inhibit the oxidizing processes. The reason for this apparent contradiction lies on the activation effect of low pH on the adenylate transfer reaction from dehydroluciferyl‐adenylate to the acceptor nucleotide.

Acyl-CoA synthetase catalyzes the synthesis of diadenosine hexaphosphate (Ap6A)

The synthesis of diadenosine hexaphosphate (Ap6A), a potent vasoconstrictor, is catalyzed by acyl-CoA synthetase from Pseudomonas fragi. In a first step AMP is transferred from ATP to tetrapolyphosphate (P4) originating adenosine pentaphosphate (p5A) which, subsequently, is the acceptor of another AMP moiety from ATP generating diadenosine hexaphosphate (Ap6A). Diadenosine pentaphosphate (Ap5A) and diadenosine tetraphosphate (Ap4A) were also synthesized in the course of the reaction. In view of the variety of biological effects described for these compounds the potential capacity of synthesis of diadenosine polyphosphates by the mammalian acyl-CoA synthetases may be relevant.

A Tridimensional Representation of Enzyme Inhibition Useful for Diagnostic Purposes

A new three dimensional representation of enzyme inhibition, applied to Lineweaver-Burk, Hanes and Eadie-Hofstee plots is presented. This type of representation has advantages for enzyme inhibition diagnosis, showing graphic characteristics that pass unnoticed in linear plots.

Enzyme inhibition as visualized with the reservoir model: Relationships between i50 and inhibition constant(s) of an enzyme inhibitor

In the reservoir model, enzyme activity is simulated by holes in the wall of a reservoir (metabolic pool) containing liquid (substrate) at a certain level (concentration). The holes are computer drawn to reflect the kinetic properties of the enzyme, namely its maximum velocity (hole area) and Km (hole position). The model is here presented as a tool to intuitively visualize the effect of the different types of reversible enzyme inhibitors on the kinetic properties of an enzyme. The relationships between the concentration of an inhibitor producing 50% inhibition of an enzyme reaction (I50) and its inhibition constant(s) are also discussed.