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

Fraga, Hugo; Silva, Joaquim C. G. Esteves da; Fontes, Rui

doi:10.1016/s0014-5793(03)00382-x

Cited by 16 publications

(17 citation statements)

References 18 publications

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“…Another consequence of luciferase's function as a ligase is the synthesis of dinucleoside polyphosphates, in a reaction analogous to the pyrophosphorolysis of L-AMP to give ATP [ Nonetheless the best conditions for the synthesis of dinucleoside polyphosphates are the opposite of those necessary for light production, namely acidic medium, while luciferase requires a physiological pH, about 7.5, to produce light (55). This contradictory effect was explained as the low pH could facilitate the transfer of the adenylate moiety of L-AMP to the acceptor (55).…”

Section: Firefly Luciferase Reactionsmentioning

confidence: 99%

Firefly bioluminescence: A mechanistic approach of luciferase catalyzed reactions

2008

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SummaryLuciferase is a general term for enzymes catalyzing visible light emission by living organisms (bioluminescence). The studies carried out with Photinus pyralis (firefly) luciferase allowed the discovery of the reaction leading to light production. It can be regarded as a two-step process: the first corresponds to the reaction of luciferase's substrate, luciferin (LH 2 ), with ATPMg 21 generating inorganic pyrophosphate and an intermediate luciferyl-adenylate (LH 2 -AMP); the second is the oxidation and decarboxylation of LH 2 -AMP to oxyluciferin, the light emitter, producing CO 2 , AMP, and photons of yellow-green light (550-570 nm). In a dark reaction LH 2 -AMP is oxidized to dehydroluciferyl-adenylate (L-AMP). Luciferase also shows acyl-coenzyme A synthetase activity, which leads to the formation of dehydroluciferyl-coenzyme A (L-CoA), luciferyl-coenzyme A (LH 2 -CoA), and fatty acyl-CoAs. Moreover luciferase catalyzes the synthesis of dinucleoside polyphosphates from nucleosides with at least a 3 0 -phosphate chain plus an intact terminal pyrophosphate moiety. The LH 2 stereospecificity is a particular feature of the bioluminescent reaction where each isomer, D-LH 2 or L-LH 2 , has a specific function. Practical applications of the luciferase system, either in its native form or with engineered proteins, encloses the analytical assay of metabolites like ATP and molecular biology studies with luc as a reporter gene, including the most recent and increasing field of bioimaging.2008 IUBMB IUBMB Life, 61(1):

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Section: Firefly Luciferase Reactionsmentioning

confidence: 99%

Firefly bioluminescence: A mechanistic approach of luciferase catalyzed reactions

2008

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“…Analysis of these mixtures by RP-HPLC allowed us to detect the substrates and products related to the Luc reactions (with absorbance maxima between 260 and 400 nm [12,20,25] and, simultaneously, detect resorufin (absorbance maxima at 571 nm) and Amplex Red (absorbance maxima at 247 nm and very long retention time).…”

Section: -Amp Is Converted Into L-ampmentioning

confidence: 99%

“…The formation of L-AMP can be directly detected when the bioluminescent reaction mixtures are supplemented with pyrophosphatase, preventing pyrophosphorolysis [see reactions (4) and (5)]. [9-12, 18, 19] The Luc-catalyzed oxidation of LH 2 -AMP into L-AMP is slower at acidic pH [20] and can represent as much as 16 % of the LH 2 consumption. [12] The importance of this oxidation and of the thiolytic splitting of L-AMP by CoA in Luc-catalyzed synthesis of dinucleoside polyphosphates [12] and on bioluminescent reaction kinetics [19] has already been studied.…”

Section: Introductionmentioning

confidence: 99%

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Firefly Luciferase Produces Hydrogen Peroxide as a Coproduct in Dehydroluciferyl Adenylate Formation

et al. 2006

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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.

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“…STPP converts a dehydroluciferyl–adenylate to a dehydroluciferin and adenosine tetraphosphate (AP 4 ) (7,11). Nucleotide (cytidine triphosphate) converts in the same way to a dinucleoside polyphosphate (7,12). Similarly, the ITP applied in this study is thought to give rise to a dehydroluciferin and adenosine tetraphosphate inosine (AP 4 I).…”

Section: Resultsmentioning

confidence: 99%

Practical application of bioluminescence enzyme immunoassay using enhancer for firefly luciferin–luciferase bioluminescence

et al. 2011

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Firefly luciferin-luciferase bioluminescence is known for its high quantum yield (41.0 ± 7.4%). Given this high quantum yield, application of this bioluminescence is expected to be useful in the field of clinical diagnostics. The kinetic profile of this bioluminescence exhibits an instant rise (<1 s) and a rapid decay in light emission (decreased to 42% after 5 s). In this study, we applied four enhancers including coenzyme A, inosine5'-triphosphate sodium salt, sodium tripolyphosphate and potassium pyrophosphate to prolong light emission. When these enhancers were used, luminescence was only decreased to 89, 83, 87 and 82% after 5 s, respectively. These materials modified the kinetic profile of bioluminescence so that the luminescence is more suitable for clinical application. It becomes more suitable because they enable highly sensitive integration and simplification of a device by separating luminescence measurements from dispensing of reagents. Using these enhancers, we then developed a bioluminescent enzyme immunoassay (BLEIA) for hepatitis B virus surface antigen (HBsAg) that employed firefly luciferase as a labeling enzyme. We compared the results obtained from the HBsAg BLEIA method with the conventional chemiluminescent enzyme immunoassay method, and found a satisfactory correlation (r=0.984, n=118).

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

Cited by 16 publications

References 18 publications

Firefly bioluminescence: A mechanistic approach of luciferase catalyzed reactions

Firefly bioluminescence: A mechanistic approach of luciferase catalyzed reactions

Firefly Luciferase Produces Hydrogen Peroxide as a Coproduct in Dehydroluciferyl Adenylate Formation

Practical application of bioluminescence enzyme immunoassay using enhancer for firefly luciferin–luciferase bioluminescence

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