Luciferyl adenylate

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. 

In the first step, luciferin is converted into luciferyl adenylate by ATP in the presence of Mg2+. In the second step, luciferyl adenylate is oxidized by molecular oxygen resulting in the emission of yellow-green light, of which the mechanism is discussed in Sections 1.1.6 and 1.1.7. Both steps, (1) and (2), are catalyzed by luciferase. The reaction of the first step is slower than that of the second step, thus the first step is the rate-limiting step. 

In the postulated bioluminescence mechanism, firefly luciferin is adenylated in the presence of luciferase, ATP and Mg2+. Luciferyl adenylate in the active site of luciferase is quickly oxygenated at its tertiary carbon (position 4), forming a hydroperoxide intermediate (A). 

Because luciferyl adenylate emitted a red chemiluminescence in the presence of base, coinciding with the red fluorescence of 5,5-dimethyloxylucferin, the keto-form monoanion Cl in its excited state is considered to be the emitter of the red light. Thus, the emitter of the yellow-green light is probably the enol-form dianion C2 in its excited state, provided that the enolization takes place within the life-time of the excited state. Although the evidence had not been conclusive, especially on the chemical structures of the light emitters that emit two different colors, the mechanism shown in Fig. 1.12 was widely believed and cited until about 1990. 

DeLuca, M., Dempsey, M. E., Hori, K., and Cormier, M. J. (1976). Source of oxygen in CO2 produced during chemiluminescence of firefly luciferyl-adenylate and Renilla luciferin. Biochem. Biophys. Res. Commun. 69 262-267. 

Rhodes, W. C., and McElroy, W. D. (1958). The synthesis and function of luciferyl-adenylate and oxyluciferyl-adenylate. J. Biol. Chem. 233 1528-1537. 

With isotopes it has been possible to show that all enzyme-catalyzed reactions are stereospecific. Before the availability of isotopes, there was no way of testing this generalization. Of course there are some apparent exceptions to prove the rule. Bently has listed a considerable number (2>, Table XIII, Chapter 6). The most interesting one to me seems to be luciferase, but that is an exception that isn t an exception. Thus, the enzyme luciferase acts on its substrate luciferin (2), in the presence of ATP and O2, to oxidize the luciferin to oxyluciferin (3). The reaction consists of an initial activation of the substrate by ATP to give luciferyl adenylate, after which the oxidation takes place. When the natural enantiomer (synthesized from D-cysteine) is activated and oxidized, light is emitted. The other enantiomer is also acted on by the enzyme, and is converted to the adenylate, but oxyluciferin is not formed, and there is no bioluminescence 37,38,38a)... 

The firefly enzyme (EC 1.13.12.7) catalyzes the intermediate formation of D(-)-luciferyl adenylate and pyrophosphate fromD(-)-luciferin and ATP, followed by the oxidative reaction of the acyl adenylate with molecular oxygen to form an enzyme-bound product in the excited... 

Each synthetase module contains three active site domains The A domain catalyzes activation of the amino acid (or hydroxyacid) by formation of an aminoacyl- or hydroxyacyl-adenylate, just as occurs with aminoacyl-tRNA synthetases. However, in three-dimensional structure the A domains do not resemble either of the classes of aminoacyl-tRNA synthetases but are similar to luciferyl adenylate (Eq. 23-46) and acyl-CoA synthetases.11 The T-domain or peptidyl carrier protein domain resembles the acyl carrier domains of fatty acid and polyketide synthetases in containing bound phos-phopantetheine (Fig. 14-1). Its -SH group, like the CCA-terminal ribosyl -OH group of a tRNA, displaces AMP, transferring the activated amino acid or hydroxy acid to the thiol sulfur of phosphopan-tetheine. The C-domain catalyzes condensation (peptidyl transfer). The first or initiation module lacks a C-domain, and the final termination module contains an extra termination domain. The process parallels that outlined in Fig. 21-11.1... 

The structure of firefly luciferin has been confirmed by total synthesis. The firefly emits a ycllow-grccn luminescence, and luciferin in this case is a benzthiazole derivative. Activation of the firefly luciferin involves the elimination of pyrophosphate from ATP widi the formation of an add anhydride linkage between the carboxyl group of luciferin and the phosphate group of adenylic acid forming luciferyl-adenylate. 

IMPORTANCE OF FIREFLY LUCIFERASE C-TERMINAL DOMAIN IN BINDING OF LUCIFERYL-ADENYLATE... 

Firefly Luciferase C-Terminal Domain in Luciferyl-Adenylate Binding... 

Dukhovich A, Sillero A, Sillero MA. Time course of luciferyl adenylate synthesis in the firefly luciferase reaction. FEBS Lett 1996 395 188-90. 

Fraga H, Esteves da Silva JCG Fontes R. Identification of luciferyl adenylate and luciferyl coenzyme A synthesized by firefly luciferase. ChemBioChem 2004 5 110-5. 

Some years ago I demonstrated that such a biochemical flash can be produced in the test tube.9,15 If oxygen is excluded from a firefly luciferase reaction mixture and then added rapidly back, a bright flash occurs, some 100 to 200 times brighter than the baseline intensity (Fig. 3). This comes from the reaction of the luciferyl adenylate active intermediate accumulated in the absence of oxygen. Note that the decay of the flash is not due to the removal of oxygen, but to the utilization of the luciferase-peroxide intermediate, so the baseline returns to a low level (Fig. 4), defined by the slow rate of reaction of ATP with luciferin. It is well known that the kinetics of firefly flashes are species specific and of functional importance in courtship communication, fixed by the rate constant for the first order decay of the peroxide intermediate formed from the adenylate. 

The first step in the formation of light in the firefly is a reaction with ATP to form luciferyl adenylate (Eq. 23-46, step The proton on the carbon may then be removed making use of the electron accepting properties of the adjacent ring system and carbonyl group before addition of the O2. The reactions should be compared to those catalyzed by oxygenases, e.g.,... 

The enzymes shows no requirement for metal ions, and there are indications that the active site is extremely hydrophobic 142). In fact, the luciferyl adenylate (58) undergoes rapid, spontaneous oxygenation in organic solvents to give a hydroperoxide (60) which eventually decomposes decarboxylatively to the oxyluciferin (57), accompanied by luminescence 142). There is no need for oxygen activation and the enzyme... 

In the initial step, the D-luciferin is activated via interaction with Mg-ATP to give the enzyme bond d-luciferyl adenylate and pyrophosphate. As shown, this reaction is reversible and therefore pyrophosphate can impede the forward reaction. Generally, only low levels of pyrophosphate are formed from the concentrations of ATP and luciferase present under normal reaction conditions. The second step is an oxidative decarboxylation which produces oxyluciferin (4) in an electronically excited state. The excited oxyluciferin returns to the ground state by the ejection of a photon (/lem = 562mn) with an impressive overall quantum yield of 0.9 (at 25°C and pH 7.8). The light intensity is directly proportional to the ATP concentration provided that the level of luciferin remains constant. 

There are a number of ATP systems currently available, including UltraSnap (Hygi-ena), PocketSwab Plus (Charm Sciences), Hy-Lite (VWR) and Clean-Trace (3M). The UltraSnap system contains a premoistened swab in a tube and the reaction reagent in a cap on the top. A certain area of surface is wiped over with the swab. Once the cap is closed the reagent moves down to the swab and the tube is shaken a few times. If there is ATP present on the swab it will react with reagent containing luciferyl adenylate. The result of the positive reaction will be bioluminescence, which can be detected in a luminometer. 

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