Luciferin-luciferase reaction scheme

The preparations of luciferin (Ln, an electron acceptor) and soluble enzyme used were crude or only partially purified. The luciferase was an insoluble particulate material, possibly composed of many substances having various functions. Moreover, the luciferin-luciferase reaction was negative when both luciferin and luciferase were prepared from certain species of luminous fungus. It appears that the light production reported was the result of a complex mechanism involving unknown substances in the test mixture, and probably the crucial step of the light-emitting reaction is not represented by the above schemes. 

The reaction scheme of Latia bioluminescence. Based on the structures of luciferin 1 (Ln) and the product of luminescence reaction 2 (OxLn), it was proposed that the luciferase-catalyzed luminescence reaction of Latia luciferin in the presence of the purple protein results in the formation of 2 moles of formic acid, as shown in the scheme A (Shimomura and Johnson, 1968c). However, when the luminescence reaction was carried out in a medium containing ascorbate and NADH (in addition to the purple protein) to increase the quantum yield, it was found that only one mole of formic acid was produced accompanied... 

Fig. 6.3.5 A reaction scheme proposed by Tsuji (2002) for the Watasenia bioluminescence. The proposed mechanism involves the adenylation of luciferase-bound luciferin by ATP, like in the bioluminescence of fireflies. However, the AMP group is split off from luciferin before the oxygenation of luciferin, differing from the mechanism of the firefly bioluminescence. Thus the role of ATP in the Watasenia bioluminescence reaction remains unclear. Reproduced with permission from Elsevier.

Of the many types of bioluminescence in nature, that of the firefly represents the most thoroughly studied and best understood biological luminescent process. The molecular mechanism of light emission by the firefly was elucidated in the 1960s in which a dioxetanone (a-peroxy lactone) was proposed as an intermediate, formed by the luciferase-catalyzed enzymatic oxidation of the firefly luciferin with molecular oxygen (Scheme 15). This biological reaction constitutes one of the most efficient luminescent processes known to date . Hence, it is not surprising that the luciferin-luciferase system finds wide use... 

Fig. 17 Reaction schemes for the bioluminescence of lucifera-ses. Both forms of luciferase catalyze the oxidation of their respective substrates (A) Renilla luciferase oxidizes coelenter-azine (B) firefly luciferase oxidizes beetle luciferin.

The luciferase from the North American firefly P. pyralis is a monomeric enzyme (62 kDa) consisting of 550 amino acid residues. The firefly luciferase produces light by the ATP-dependent oxidation of D(-)-luciferin (LH2) (see Scheme 8.3). The reaction involves an enzyme-hound luciferyl adenylate intermediate. The peak light emission occurs at 562 nm (yellow—green, quantum yield of 0.88) in dianionic form between pH 7 and 8 or at 610 nm (red, quantum yield of 0.2) in monoanionic form at pH values below 7. The red shift also occurs in the presence of Zn (2.3 mM) or Cd (12 mM) (58). 

Luciferin includes a whole family of compounds whose heterocyclic structures vary fix>m one organism to another. Most luciferase enzymes use the same cofactors as metabolic processes (ATP, FMN, NADH), and bioluminescence is easily associated with other types of Inological reactions for analytical applications. Firefly ludferase is used tt> follow processes that use adenosine triphosphate (ATP) as a cofactor, for example, the measurement of biomass, the detection of a bacterial infection, antibiotic assays, and the monitoring of other enzymatic reactions that consume or produce ATP. Luciferase catalyses all these reactions according to the following overall reaction scheme ... 

The following schemes represent the overall reaction of firefly bioluminescence (McElroy and DeLuca, 1978), where E is luciferase LH2 is D-luciferin PP is pyrophosphate AMP is adenosine phosphate LH2-AMP is D-luciferyl adenylate (an anhydride formed between the carboxyl group of luciferin and the phosphate group of AMP) and L is oxyluciferin. 

Studies with Cypridina (154), Oplophorus 150) and Renilla 155) luciferases have uncovered the following steps (Scheme 26) 156). First, the appropriate luciferase deprotonates the luciferin (62) to its tetrahedral carbanion (64). Reaction of the latter with oxygen yields the hydroperoxide (65). Cyclization affords the so-called high energy intermediate, the dioxetanone (66), which on cleavage loses carbon dioxide to give the amide (67) accompanied by chemiluminescence. 

If luminescence is a result of a biochemical reaction, the principle is called bioluminescence. The most frequently used bioluminescence system is that of the firefly. The enzyme luciferase catalyses the oxidation of luciferin as a substrate in the presence of adenosine triphosphate (ATP) (Scheme 7). Another bioluminescence system makes use of a luciferase from certain marine bacteria. A long-chain aldehyde is oxidized in the presence of luciferase, an oxido-reductase and NAD/NADH. Recently, a photoprotein isolated from the bioluminescent jellyfish Aequorea victoria, has been found to be an efficient bioluminescence label for immunoassays. 

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