Báo cáo khoa học: Coenzyme A affects firefly luciferase luminescence because it acts as a substrate and not as an allosteric effector pot

Coenzyme A affects firefly luciferase luminescence because it acts as a substrate and not as an allosteric effector Hugo Fraga 1,2 , Diogo Fernandes 1,2 , Rui Fontes 2 and Joaquim C. G. Esteves da Silva 1 1 Departmento de Quı ´ mica, Faculdade de Cie ˆ ncias da Universidade do Porto, Portugal 2 Servic¸o de Bioquı ´ mica (U38-FCT), Faculdade de Medicina da Universidade do Porto, Portugal Firefly luciferase (LUC, EC 1.3.12.7) is an enzyme that catalyses the oxidation of luciferin (LH 2 ), in the pres- ence of ATP and Mg 2+ , giving rise to light [1,2]. The bioluminescence reaction involves the reaction of LH 2 and ATP to form luciferyl-adenylate (LH 2 -AMP) (Reaction 1 and Fig. 1). LH 2 -AMP is then oxidized by molecular oxygen and, through a series of inter- mediates, gives rise to AMP, inorganic pyrophosphate (PPi), CO 2 and oxyluciferin (Reaction 2 and Fig. 1), the presumed light emitter [2]. LUC þ LH 2 þ ATP Ð LUCÆLH 2 -AMP þ PPi ð1Þ LUCÆLH 2 -AMP þ O 2 À! LUC þ AMP þ CO 2 þ oxyluciferin þ photon ð2Þ An ATP determination assay based on LUC biolumin- escence reaction is an important analytical tool, mainly Keywords Coenzyme A; dehydroluciferyl-adenylate; dehydroluciferyl-coenzyme A; dephospho- coenzyme A; firefly luciferase Correspondence J. C. G. Esteves da Silva, Departmento de Quı ´ mica, Faculdade de Cie ˆ ncias da Universidade do Porto, R. Campo Alegre 687, 4169–007 Porto, Portugal Fax: +351 226082959 Tel: +351 226082869 E-mail: jcsilva@fc.up.pt (Received 24 May 2005, revised 28 June 2005, accepted 18 July 2005) doi:10.1111/j.1742-4658.2005.04895.x 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 (LH 2 -AMP), and is a powerful inhibitor of the bioluminescence reaction (IC 50 ¼ 6nm). 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 dehydro- luciferyl-CoA (L-CoA), a much less powerful inhibitor (IC50 ¼ 5 lm). Moreover, the V max for L-CoA formation was determined as 160 min )1 , which is one order of magnitude higher than the V max of the biolumines- cence 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 anta- gonize 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 dehydro- luciferyl-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 biolumin- escence reaction, the fraction of LH 2 -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. Abbreviations dephospho-CoA, dephospho-coenzyme A; dethio-CoA, dethio-coenzyme A; L, dehydroluciferin; L-AMP, dehydroluciferyl-adenylate; LUC, firefly luciferase; L-CoA, dehydroluciferyl-coenzyme A; L-dephospho-CoA, dehydroluciferyl-dephospho-coenzyme A; LH 2 , firefly luciferin; LH 2 -AMP, luciferyl-adenylate; LH 2 -CoA, luciferyl-coenzyme A; PPase, inorganic pyrophosphatase; PPi, inorganic pyrophosphate; RLU, relative light units. 5206 FEBS Journal 272 (2005) 5206–5216 ª 2005 FEBS because of its high sensitivity and specificity [3]. Com- mercial ATP assay kits, apart from LH 2 and LUC, contain coenzyme A (CoA), which modifies the kinetic profile making it more suitable for analytical work: instead of a flash profile (high light emission when the reaction starts and a rapid decay into a low basal level) a stable and prolonged light production is obtained [4–9]. However, despite its widespread use, the explanation for its effect remains unclear. In 1958, Airth, Rhodes and McElroy suggested that CoA was able to remove oxyluciferyl-adenylate from the enzyme core, forming oxyluciferyl-CoA. Consistent with this idea, oxyluciferyl-adenylate was identified as a product and a potent inhibitor of the bioluminescence activity [4]. The chemical structure of oxyluciferin was determined years later [10] and it is now known that the compound named by Airth, Rhodes and McElroy as oxyluciferyl-adenylate is, actually, dehydroluciferyl- adenylate (L-AMP) [11,12]. This compound and dehydro- luciferin (L) are side products of the bioluminescence reaction [11–13]; L-AMP is formed from dehydrogena- tion of LH 2 -AMP and L results from the pyrophos- phorolysis of L-AMP (Reaction 3 and Fig. 1). LUCÆL-AMP þ PPi Ð LUC þ L þ AMP ð3Þ L-AMP is a potent inhibitor of the bioluminescence reaction and its thiolysis by CoA (Reaction 4 and Fig. 1) [14] is one of the explanations for the light sta- bilizing effect of CoA [4,11,12,15,16]. LUCÆL-AMP þ CoA Ð LUC þ L-CoA þ AMP ð4Þ Apart from this thiolytic activity based mechanism, it was suggested that the effect of CoA and other CoA analogues might be explained by an allosteric confor- mation change that enhanced product removal [7]. This was supported by the observation of a light acti- vator effect of compounds presumably unable to react with L-AMP, namely dephospho-CoA and acetyl- CoA. In this work, we have investigated the inhibitory effects of chemically synthesized L-AMP [17] and dehydroluciferyl-CoA (L-CoA) [18] on light produc- tion and the role of CoA and diverse CoA analogues as antagonists of the inhibitory effect of L-AMP. The main conclusion is that the effect of CoA on firefly luciferase bioluminescence is not allosteric but, instead, is due to the LUC-catalysed thiolytic split of L-AMP into L-CoA, as earlier postulated. Results and Discussion Preliminary results Confirming results of other authors [4–9], we observed that when CoA was supplemented to LUC biolumines- cence reaction mixtures it prevented the rapid decay of light production (Fig. 2). In agreement with the obser- vations of some authors [4,6,8,9] and in contradiction with others [5,7], in the conditions used in this work, i.e. when light production was initiated by injecting a mixture of ATP and LH 2 into a solution containing LUC, we did not observe a marked effect of CoA on the maximum intensity of bioluminescence (Fig. 2). The extent of stabilization of the light output along the assay time depended on the concentrations of ATP and LH 2 used. Actually, for a fixed concentration of Fig. 1. LUC catalyzed reactions. In the presence of ATP, LH 2 is activated to LH 2 -AMP, which, through a series of intermediates, is oxidized by O 2 giving rise to oxyluciferin, CO 2 and AMP. In a side reaction LH 2 -AMP is oxidized to L-AMP; molecular oxygen is presumed to be the oxidant but the nature of the reduced product is unknown. L-AMP can be split by PPi (pyrophosphorolysis) or by CoA (thiolysis). H. Fraga et al. Coenzyme A and luciferase bioluminescence FEBS Journal 272 (2005) 5206–5216 ª 2005 FEBS 5207 LUC, the decay without CoA and the stabilizing effect of CoA were more pronounced when high concentra- tions of ATP and LH 2 were used (Fig. 2). These results confirmed the idea that the light production decay is due to formation of a product (or products) that inhibit the bioluminescent reaction and that CoA, somehow, antagonizes that inhibition. At the time the maximum intensity was attained (1–2 s) the formation of the inhibitory product antagonized by CoA has only began and the effect of CoA at that assay time was nil or a discrete activation (always less than 20%). Fontes et al. [11] observed that CoA could antagonize the inhibitory effect of chemically synthesized L on light production. This CoA effect was explained by the thio- lytic split of L-AMP that gives rise to L-CoA, which was presumed not to be an inhibitor. In that work, it was assumed that L-AMP was the true inhibitor and that it was formed by ATP-dependent adenylation of the added L. In the course of the bioluminescence reac- tion, L-AMP is formed directly from the intermediate LH 2 -AMP (Fig. 1) [11]. Therefore, the addition of chemically synthesized L-AMP would be a good mimic of its formation in the enzyme core. Pre-incubating LUC with L-AMP and starting the light reaction by injecting a mixture containing LH 2 and ATP supplemented with CoA, we observed a marked antagonizing effect of CoA on the inhibitory effect of L-AMP – in Fig. 3 the results obtained with 0.5 lm L-AMP are shown. The flash height increased with the concentration of CoA; at this concentration of L-AMP, Fig. 2. The stabilizing effect of CoA on firefly luciferase bioluminescence. Mixtures containing ATP and LH 2 were injected into other mixtures containing Hepes, MgCl 2 , and LUC (20 nM) supplemented (solid symbols) or not supplemented (open symbols) with CoA (50 lM). All the indicated quantities are final concentrations. Fig. 3. Activator effect of CoA and dephospho-CoA on L-AMP inhib- ited luciferase bioluminescence.The light production assays were performed in the presence of 0.5 l M L-AMP that was preincubated with LUC (60 n M) for half a minute. The light reaction was initiated by the injection of a mixture containing LH 2 (10 lM) and ATP (50 l M), supplemented with the indicated concentrations of CoA (solid diamonds) dephospho-CoA (solid squares) or dethio-CoA (solid circles). The discontinuous line and the open diamonds repre- sent the result obtained in the absence of L-AMP and in the pres- ence of the indicated concentrations of CoA. All the indicated quantities are final concentrations. Coenzyme A and luciferase bioluminescence H. Fraga et al. 5208 FEBS Journal 272 (2005) 5206–5216 ª 2005 FEBS the flash height obtained with 100 lm CoA (the highest concentration used) was only 13% lower than the flash height of the control without L-AMP. The powerful antagonizing effect of CoA on the inhibitory action of L-AMP supports the thiolytic mechanism. Evaluation of the thiolytic activity based mechanism The thiolytic mechanism would be further supported if L-CoA was, indeed, a less powerful inhibitor than L-AMP. To test this hypothesis, we studied the bio- luminescent reaction in the presence of different concen- trations of chemically synthesized L-CoA (0–243 lm). The inhibitory effect of L-AMP (0–2.2 lm) was also tested in experiments performed under similar condi- tions (6 nm LUC) and the results obtained confirmed our hypothesis: the IC50 of L-AMP (6 nm) was three orders of magnitude lower than that for L-CoA (5 lm). The thiolytic mechanism was also supported by the fact that the inhibitory effect of L-CoA was not reversed by CoA (Fig. 4). When the concentrations of the inhibitors were 4 times their respective IC50 (that is, 24 nm for L-AMP and 20 lm for L-CoA) the degree of activation (as defined in Fig. 4) induced by the supplementation of the reaction mixtures with 100 lm CoA was 8 in the case of L-AMP, and nil in the case of L-CoA (Fig. 4). Using RP-HPLC we have confirmed that, as expected, L-CoA did not react with CoA. However, until now, the velocity of thiolytic reaction was not considered. When CoA was injected into assay mixtures, where LUC had been producing light (and L-AMP) for 1 min, we observed a second flash (Fig. 5). When the bioluminescence reaction was taking place in the presence of added L-AMP, light flashes were also observed at the time of CoA injection (Fig. 5). If these flashes resulted from the thiolytic removal of the LUC produced L-AMP (Fig. 5A) or the thiolytic removal of the added L-AMP (Fig. 5B,C) from the enzyme core, these reactions had to very fast. As the time to attain the new maximum velocity was less than 2 s, this should be the time for the LUC catalysed removal of the L-AMP from the enzyme core. RP-HPLC based experiments were designed to determine the velocity of the thiolytic split of L-AMP by CoA. These experiments confirmed that this reac- tion was indeed very fast (Fig. 6A). The incubation of 30 lm L-AMP with various concentrations of CoA and LUC allowed us to estimate the V max for L-CoA formation as 160 min )1 . This velocity is one order of magnitude higher than the V max for the wild type LUC catalysed light production reported by Branchini et al. [8,19]. The numbers reported by the group of Branchini (8–14 min )1 ) were calculated performing the bioluminescent reaction in a calibrated luminometer that allows the measurement of real time photon emis- sion [19]. From RP-HPLC literature results (Fontes et al. [12], Fig. 4) we calculated that the average velo- city of LH 2 transformation into non-L-AMP and non- L products in the first 15 s of reaction was 2 min )1 .As the V max values reported by Branchini et al. [8,19] were calculated from maximal light intensities at 0.5 s inte- gration time, considering the flash profile of the light reaction, it is reasonable to consider that the numbers obtained with these two different methods agree. Thus, both these results validate the idea that the thiolytic reaction can be faster than the bioluminescence reaction. According to Oba et al. [20], the values of V max for LUC catalysed formation of linolenyl-CoA (from ATP, linolenic acid and CoA) and for light production are similar. As we studied the synthesis of L-CoA from L-AMP and CoA, bypassing the adenylation step, it was not a big surprise to find out that the reaction of synthesis of L-CoA could be faster than the light production reaction. Fig. 4. Effect of CoA on bioluminescent reactions inhibited by L-AMP or L-CoA.The light production was initiated by coinjecting LH 2 and ATP supplemented or not supplemented with CoA (100 l M) into solutions where L-AMP or L-CoA was preincubated with LUC (6 n M) for 1 min. All the indicated quantities are final con- centrations. In parentheses we show the degree of activation that was calculated using the formula (vCoA-vi) ⁄ vi. vi is the maximum RLU observed in the presence of the indicated inhibitor (L-AMP or L-CoA) and in the absence of CoA; vCoA is the maximum RLU when both inhibitor and CoA were present. The bar corresponding to 20 l M L-AMP in the absence of CoA is too low to be represen- ted in the scale of the figure (flash height of 196 RLU). H. Fraga et al. Coenzyme A and luciferase bioluminescence FEBS Journal 272 (2005) 5206–5216 ª 2005 FEBS 5209 Study of the effect of CoA analogues Despite the previous observations, we could not dis- card completely the allosteric mechanism proposed by Ford et al. [7] as an alternative explanation for the effect of CoA. As already mentioned, compounds not expected to react with L-AMP were shown to stabilize light production [7]. The literature reports about the effect of dephospho-CoA (a CoA analogue lacking a terminal phosphate on position 3¢) on light were apparently contradictory. Ford et al. [7] found that dephospho-CoA supplementation of bioluminescent reaction mixtures stabilized the light production, whereas Airth et al. [4] reported that, when it was added to bioluminescence reaction mixtures that have already produced light for 3 min, it had no effect. As a first approach, we studied the effect of dephos- pho-CoA as a possible antagonist of L-AMP on light production finding that, although with lower potency, it mimicked the CoA effect (Fig. 3). When it was injec- ted into L-AMP supplemented bioluminescent reac- tions, the levels of light production attained, although lower than those reached with added CoA, represented significant activations (Fig. 5B,C). When injected into non supplemented bioluminescent reaction mixtures that have produced light (and L-AMP) for 1 min, the most obvious difference between the effect of CoA and the effect of dephospho-CoA was the onset time: CoA produced a fast second flash, while dephospho-CoA produced a slow rise of the light intensity that took 10 s to reach a maximum (Fig. 5A). Acetyl-CoA was studied in parallel and the results obtained were similar to those reported for dephospho-CoA (not shown). Ford et al. [7] showed that dethio-CoA had no effect as light stabilizer and our group confirmed that it was unable to react with L-AMP [21]. In Figs 3 and 5, we show that dethio-CoA had no effect as an antagonist of L-AMP inhibition. Interpreting the absence of effect of dethio-CoA, Ford et al. emphasized the importance of the thiol group for the recognition of the CoA putative allosteric site [7]. However, it is difficult to accept that an allosteric site could recognize acetyl- CoA, dephospho-CoA and not a CoA analogue (dethio- CoA) with greater structural resemblance to CoA. To pursue this investigation, RP-HPLC was used to study the reactivity of dephospho-CoA and acetyl- CoA with L-AMP. When dephospho-CoA was incuba- ted with L-AMP in the presence of LUC, we detected the formation of a new compound. In Fig. 7, the chromatographic peak corresponding to the compound formed from dephospho-CoA and L-AMP by LUC (peak 1) has an absorbance spectrum identical to the one of L-CoA [18] but with a longer retention time. It has been reported that acyl-CoA synthetases can thio- esterify fatty acids using dephospho-CoA instead of CoA [22,23] and the functional and structural similarity between LUC and acyl-CoA synthetases are also well known [1,15,17,18,20,21,24,25]. With this background, we suspected that the new compound formed was dehy- droluciferyl-dephospho-CoA (L-dephospho-CoA) and Fig. 5. Effect of CoA and CoA analogues injection on L-AMP inhibited light production.The light reaction was intiated (0 time) in a volume of 50 lL by the addition of LUC (60 n M) to a mixture containing Hepes, MgCl 2 ,LH 2 and ATP and different concentrations of L-AMP (zero in A, 0.5 l M in B and 10 lM in C). After 60 s, 50 lL of a solution (100 lM) of CoA (diamonds; uppermost curve), dephospho-CoA (squares), dethio- CoA (circles) or water (continuous line) was injected. All the indicated quantities are final concentrations. Coenzyme A and luciferase bioluminescence H. Fraga et al. 5210 FEBS Journal 272 (2005) 5206–5216 ª 2005 FEBS we confirmed our hypothesis taking advantage of the ability of alkaline phosphatase to hydrolyse terminal phosphates. When L-CoA was treated with this enzyme, RP-HPLC analysis of the reactions mixtures revealed the disappearance L-CoA and the appearance of a new peak; this peak had spectrum and retention time equal to the compound formed by LUC from dephospho-CoA and L-AMP (not shown). In Fig. 6 the LUC thiolytic activities with CoA and dephospho- CoA and the degree of activation caused by the same compounds on L-AMP inhibited bioluminescence are compared. The correlation between the thiolytic activit- ies and the degrees of activations induced is remark- able. The apparent K m of CoA in the thiolytic reaction and the apparent K a of the same compound in light production, both determined at the same fixed concentration of L-AMP (30 lm) were very similar (76 and 73 lm, respectively). It could be concluded that the lower potency of dephospho-CoA as antagonist of L-AMP inhibition (Figs 2,4 and 5) and the slow rise observed when it was injected into reaction mixtures that have produced L-AMP for 1 minute (Fig. 4A) was a consequence of its slower reactivity with L-AMP. In the case of acetyl-CoA, however, some doubt remained. This compound antagonized the inhibitor effect of L-AMP, but it has no free thiol group and therefore is unable to react with L-AMP. In reaction mixtures where L-AMP and acetyl-CoA were incuba- ted in the presence of LUC, we could observe the for- mation of L-CoA (Fig. 7). The most obvious explanation for the formation of L-CoA in those con- ditions was the presence of contaminant CoA in the commercial acetyl-CoA preparation used. Actually, the Fig. 7. RP-HPLC analysis of reaction mixtures containing L-AMP, LUC, CoA and CoA analogues. Reaction mixtures containing L-AMP (20 l M), LUC, and CoA or the indicated CoA analogues were incu- bated for 10 min. After stopped by the addition of methanol the reaction mixtures were centrifuged and the supernatants analysed by RP-HPLC as referred to in the Experimental procedures section. Fig. 6. Effect of the concentration of CoA and dephospho-CoA on the velocity of formation of L-CoA and L-dephospho-CoA (A) and on L-AMP inhibited light production (B). The velocities of formation of L-CoA (diamonds) or L-dephospho-CoA (squares) were studied ana- lyzing by RP-HPLC reaction mixtures containing 30 l M L-AMP, the indicated concentrations of CoA or dephospho-CoA, Hepes, MgCl 2 and LUC. The effect of CoA and dephospho-CoA on L-AMP inhib- ited light production was studied in a luminometer coinjecting the same compounds with LH 2 and ATP. The degree of activation has been defined in Fig. 4. H. Fraga et al. Coenzyme A and luciferase bioluminescence FEBS Journal 272 (2005) 5206–5216 ª 2005 FEBS 5211 contamination of commercial preparations of acetyl- CoA with CoA was already reported by Ford et al. [7]. To confirm our suspicions, we converted the resi- dual CoA present in the commercial acetyl-CoA into acetyl-CoA preincubating it with ATP, acetate, MgCl 2 , acetyl-CoA synthetase and inorganic pyrophosphatase (PPase). Then, we confirmed that treated acetyl-CoA was no longer antagonist of L-AMP in bioluminescent reactions (data not shown). So, the antagonizing effect of acetyl-CoA over L-AMP light production inhibition was due to the presence of contaminant CoA. At this stage, we could conclude that all the anta- gonists of L-AMP inhibition tested were substrates of LUC promoting the thiolytic split of L-AMP (Fig. 7) and that a clear positive correlation between the two phenomena existed (Fig. 6). These results constitute clear evidence in support of the idea that the thiolytic split of L-AMP is an essential condition for the activa- tor effect observed when L-AMP was added to or had been produced in bioluminescent reaction mixtures. The role of L-AMP produced by LUC on light decay Although the experimental evidence is scarce [26], or even nonexistent [27], oxyluciferin (referred to as ‘the product’) is frequently referred as the compound that causes the inhibition that induces the premature light decay [7,14,28–30]. The allosteric mechanism proposed by Ford et al. [7] to explain the stabilizing effect of CoA was in line with the ideas that were generally accepted at the time their work was undertaken. Actually, oxy- luciferin has no carboxylic group [10,13] and it is pre- sumed that it cannot react with CoA. The possibilities that AMP, another product formed in the biolumines- cence reaction, can have a role either in light decay or in the effect of CoA are even weaker: apart from the absence of a carboxylic group it has been shown that it is a very weak inhibitor (K i ¼ 240 lm) [31]. Trying to get some insight into the factors that cause the light decay and into the relative importance of L-AMP and other possible inhibitors formed in the course of the bioluminescence reaction, we studied the way different concentrations of LH 2 (10 or 60 lm) and ATP (10 or 150 lm) affected the decay and the effect of injecting CoA after 1 minute of incubation. In order to exclude the interference of the PPi produced, the experi- ments were performed in the presence and in the absence of PPase (Fig. 8). As expected, the decay was more pro- nounced when higher concentrations of ATP and LH 2 were used and even more pronounced when PPase was simultaneously present. PPase hydrolyses PPi that, when Fig. 8. Role of L-AMP produced in the course of bioluminescent reaction on the light decay. Mixtures of ATP and LH 2 were injected (60 lL) into assay tubes containing 90 lL of a solution of Hepes, MgCl 2 and LUC (20 nM) supplemented (solid symbols) or not supplemented (open symbols) with PPase (1 lg of protein per mL). At 1 min of incubation 30 lL of CoA (50 l M) was injected. All the indicated quantities are final concentrations. Coenzyme A and luciferase bioluminescence H. Fraga et al. 5212 FEBS Journal 272 (2005) 5206–5216 ª 2005 FEBS produced in sufficient quantity, that is, at high light out- put (Reactions 1 and 2), can cause pyrophosphorolytic removal of produced L-AMP (Reaction 3). When the concentrations of LH 2 and ATP were low (both 10 lm; Fig. 8) the velocity of light production decreased 65% in the first minute of reaction and CoA injection had only a modest effect on light production velocity. So, we could conclude that, under these con- ditions, most of the inhibitory effect was due to prod- ucts or intermediates that cannot be antagonized by CoA. However, at higher LH 2 and ⁄ or ATP concentra- tions most of the first minute light decay could be anta- gonized by CoA injection. At 150 lm ATP and 60 lm LH 2 (Fig. 8), for example, the light decays more than 90% in the first minute of reaction but most of that inhibition could be antagonized by CoA; that is, only a small part (about 20%) of the inhibition developed in the first minute of assay was due to the formation of compounds that could not be antagonized by CoA. If we accept that L-AMP is the only product whose inhib- itory effect can be antagonized by CoA, we should con- clude that the fraction of L-AMP formation, relative to other inhibitors, increases when the concentrations of LH 2 or ATP increases. Conclusions Although the stabilizing effect of CoA on firefly bio- luminescence has been known since 1958, the respon- sible mechanism remained controversial. As CoA is not directly involved in the chemistry of light produc- tion per se, an allosteric effect on luciferase has been frequently put forward as a sensible explanation for the observed phenomenon. Actually, we have found that the activator effect of CoA on L-AMP inhibited firefly luciferase bioluminescent reaction is so fast that it mimics an allosteric effect. However, we have also demonstrated that the mechanism behind the CoA effect is not allosteric, involving, instead, a rapid thio- lytic reaction that splits L-AMP, a strong inhibitor formed as a side product in the bioluminescence reac- tion. We do not deny that conformation changes can also be involved in the CoA effect: it has been pro- posed, more than 40 years ago [32], that the binding of substrates to enzymes, the reactions in the enzyme core and the release of the products imply induced fit chan- ges in the enzyme conformation. Apart from the allosteric mechanism proposed by Ford [7], another mechanism to explain the stabilizing effect of CoA has also been formulated. It suggests that a reaction between the intermediate d-LH 2 -AMP and CoA, giving rise to luciferyl-CoA (LH 2 -CoA), might have a role on the stabilizing effect under discussion [1]. However, weakening this hypothesis it has already been shown that this reaction is very slow (less than 0.1 min )1 ) and only occurs under anaerobic conditions [16,21]. Accordingly, the production of light from LH 2 -CoA and AMP depends on very high con- centrations of LUC and AMP [33]. We are aware that the LUC catalysed synthesis of L-AMP is not the only reason for the flash profile of the bioluminescent reaction. Our experimental work seems to show that, when low concentrations of LH 2 and ATP are used, the fraction of LH 2 -AMP that is oxidized into L-AMP is lower and the importance of non-L-AMP inhibitory products and ⁄ or intermediates in the light decay is higher. In this work, it has also been shown that luciferase can be more efficient as a catalyst in the thiolytic split of L-AMP than as a light producing enzyme. Consid- ering the structural similarity between firefly luciferase and acyl-CoA synthetases, our achievement is not as strange as it seems to be and supports the theory that nowadays firefly luciferase evolved from an ancestral acyl-CoA synthetase [1]. Given the luciferase catalysed reactivity of CoA, the CoA binding site should be seen as part of luciferase active centre. Presently, there are many other enzymes containing known allosteric sites that may have evolved from ancestral nonallosteric enzymes. Our results suggest that, at least in some cases, nowadays allosteric sites may correspond to part of the active centre of ancestral nonallosteric enzymes. Moreover, as was the case of firefly luciferase, it is possible that, under certain experimental conditions, these allosteric sites may show functional activity as enzyme active sites. Experimental procedures A stock solution of commercial LUC (L9506) purchased from Sigma (St Louis, MO, USA) was prepared by dissol- ving the lyophilized powder in 0.5 m Hepes pH 7.5 (15 mg lyophilisate per mL; 60 l m LUC). Stock solutions of ace- tyl-CoA synthetase alkaline phosphatase, and PPase (all Sigma; A1765, P7923 and I1891, respectively) were pre- pared by dissolving the lyophilized powders in water to 1.25, 0.23 and 0.1 mg of protein per mL, respectively. All the enzyme stock solutions were stored at )20 °C. LH 2 , ATP, CoA, dephospho-CoA, acetyl-CoA, dethio-CoA and Hepes were purchased from Sigma. Ethyl chloroformate and 2-cyano-6-methoxybenzothiazole were purchased from Aldrich (Steinheim, Germany) and triethylamine was purchased from Fluka (Buchs, Switzerland). L, L-AMP were chemically synthesized as described pre- viously [17,34,35]. L-CoA was chemically synthesized in a straightforward adaptation of the method employed to H. Fraga et al. Coenzyme A and luciferase bioluminescence FEBS Journal 272 (2005) 5206–5216 ª 2005 FEBS 5213 obtain LH 2 -CoA synthesis [21] and the chemical characteri- zation was performed as described previously [18]. Desalt- ing of the RP-HPLC purified L-CoA was achieved employing a reverse phase C18 extraction cartridge [Lichro- lut RP-18 (40–63 lm), Merck, Darmstadt, Germany] and the phosphate content of the desalted solution was verified by a variation of the molybdate method [36]. All the enzyme reactions took place at ambient tempera- ture (24–27 °C) and were performed at least in duplicate. Luciferase catalysed light production assays The bioluminescence tests were performed in a homemade luminometer using a Hamamatsu HCL35 photomultiplier tube (Middlesex, NJ, USA). Unless otherwise indicated, the light reaction was initiated by the injection of 50 lLofa mixture of ATP (50 l m) and LH 2 (10 lm) supplemented or not with CoA and CoA analogues (0–600 lm) into a trans- parent assay tube containing 50 lL of another mixture: Hepes pH 8.2 (50 mm), MgCl 2 (2 mm) and LUC (6– 120 lm). This last mixture could, in some experiments, be supplemented with L-AMP, L-CoA or CoA. The indicated quantities are final concentrations. The light was integrated and recorded in 1 s intervals. When the light production was too high (1 mm ATP) a 1% filter that reduces the light reaching the photomultiplier tube was used. Effect of acetyl-CoA treated with acetyl-CoA synthetase on the bioluminescent reaction A reaction mixture containing in a final volume of 250 lL, 75 lm ATP, 50 mm Hepes pH 8.2, 1 mm MgCl 2 , 300 lm acetic acid, 1.5 mm commercial acetyl-CoA, PPase (2 lgof protein per mL) and acetyl-CoA synthetase (50 lg of protein per mL) was preincubated at ambient temperature. At differ- ent times of preincubation (0–20 min), 25 lL aliquots were withdrawn and added to transparent tubes that already con- tained 25 lL of a mixture of MgCl 2 , Hepes pH 8.2, L-AMP and LUC. The light reaction was initiated by injecting 50 lL of a mixture containing 20 lm LH 2 and 300 lm ATP. After the injection the concentrations of MgCl 2 (2.25 mm), Hepes (62.5 mm), L-AMP (10 lm) and LUC (120 nm) were the indicated in parenthesis. A control assay with all compo- nents except commercial acetyl-CoA was also performed. RP-HPLC analysed luciferase assays To study the reactivity of L-AMP with CoA and commer- cial CoA analogues the following procedure was used. The reaction mixtures contained Hepes pH 8.2 (100 mm), MgCl 2 (4 mm), CoA, dethio-CoA, dephospho-CoA or acetyl- CoA (all of them 200 lm), L-AMP (20 lm) and LUC (0.12 lm when CoA and dephospho-CoA were used and 2.4 lm in the other cases). After 10 min of incubation, the enzyme reactions were stopped by the addition of one volume of a solution of 66% of methanol, centrifuged for 2 min at 13 000 g and the supernatant injected (20 lL) into the RP-HPLC column. The eluent used was an aqueous solution of 32% methanol and 2.9 mm phosphate buffer (pH 7.0); the flux rate was set to 1.1 mLÆmin )1 . The chro- matographic system was constituted by a HP-1100 isocratic pump, a Rheodyne manual injection valve, a Chromolith C18 column (Merck) and a Unicam Crystal 250 UV-Vis diode array detector. For the identification of L-dephospho-CoA an assay con- taining CoA (200 lm), L-AMP (40 lm), LUC (0.6 lm), Hepes pH 8.2 and MgCl 2 was incubated for 5 min and then treated for 15 min with alkaline phosphatase (1.2 lgof protein per mL). This mixture was then stopped with one volume of a solution of 66% methanol and analysed by RP-HPLC as described above. A similar procedure was applied to chemically synthesized L-CoA. To discard the possibility that LUC catalyses any reaction between L-CoA and CoA, these compounds were added (to final concentrations of 20 and 100 l m, respectively) into assay tubes that contained Hepes pH 8.2, MgCl 2 and LUC (60 nm) and after 30 s, 5 and 10 min of incubation, aliquots were withdraw and analysed as described above. Effect of CoA and dephospho-CoA concentrations on the thiolytic reaction The effect of the concentration of CoA and dephospho- CoA on the rate of the thiolytic reaction was determined measuring the rate of L-CoA and L-dephospho-CoA forma- tion, respectively. The reaction mixtures contained in a final volume of 120 lL: 30 l m L-AMP, 50 mm Hepes pH 8.2, 2mm MgCl 2 , 0–600 lm CoA or dephospho-CoA and 120 nm LUC. The reactions were initiated with LUC addi- tion and, at 30 s, 3 and 6 min of incubation, 35 lL aliquots were withdrawn. Except for the phosphate buffer concentra- tion in the eluent and the flux rate (which was 4.9 mm and 1mLÆmin )1 , for the case of CoA, and 2 mm and 1.7 mLÆ min )1 , for the case of dephospho-CoA), the aliquots were analysed as described above. In parallel, luminometer based assays were performed in similar conditions but, in these assays, light was produced because LH 2 (10 lm) and ATP (50 lm) were coinjected with CoA or dephospho-CoA. Acknowledgements Financial support from Fundac¸ a ˜ o para a Cieˆ ncia e Tecnologia (Lisboa) (FSE-FEDER) (Project POCTI ⁄ QUI ⁄ 37768 ⁄ 2001) (PhD grant SFRH ⁄ BD ⁄ 1395 to Hugo Fraga) is acknowledged. We also acknowledge Programa Cieˆ ncia Viva (Diogo Fernandes) and Abel Duarte (Instituto Superior de Engenharia do Porto) for his help in the construction of the luminometer. Coenzyme A and luciferase bioluminescence H. Fraga et al. 5214 FEBS Journal 272 (2005) 5206–5216 ª 2005 FEBS References 1 Wood KV (1995) The chemical mechanism and evolu- tionary development of beetle bioluminescence. Photo- chem Photobiol 62, 662–673. 2 Rhodes WC & McElroy WD (1958) The synthesis and function of luciferyl-adenylate and oxyluciferyl-adenyl- ate. J Biol Chem 233, 1528–1537. 3 Strehler BL & McElroy WD (1957) Assay of adenosine triphosphate. Methods Enzymol 3, 871–873. 4 Airth RL, Rhodes WC & McElroy WD (1958) The function of coenzyme A in luminescence. Biochim Biophys Acta 27, 519–532. 5 Wood KV (1990) Novel assay of firefly luciferase provi- ding greater sensitivity and ease of use. J Cell Biol 111, 380a. 6 Pazzagli M, Devine JH, Peterson DO & Baldwin TO (1992) Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells. Anal Biochem 204, 315–323. 7 Ford SR, Buck LM & Leach FR (1995) Does the sulfhydryl or the adenine moiety of CoA enhance firefly luciferase activity? Biochim Biophys Acta 1252, 180–184. 8 Branchini BR, Southworth TL, Murtiashaw MH, Wil- kinson SR, Khattak NF, Rosenberg JC & Zimmer M (2005) Mutagenesis evidence that the partial reactions of firefly bioluminescence are catalyzed by different conformations of the luciferase C-terminal domain. Biochemistry 44, 1385–1393. 9 Wang CY & Andrade JD (1996) Surfactants and coenzyme A as cooperative enhancers of the activity of firefly luciferase. In Bioluminescence and Chemiluminescence: Molecular Reporting with Photons. Proceedings of 9th International Symposium (Hastings JW, Kricka LJ & Stanley PE, eds), pp. 253–256. John Wiley, Chichester, UK. 10 Suzuki N & Goto T (1971) Firefly bioluminescence II. Identification of 2-(6¢-hydroxybenzothiazol-2¢-YL)- 4-hydroxythiazole as a product in the bioluminescence of firefly lanterns and as a product in the chemilumines- cence of firefly luciferin in DMSO. Tetrahedron Lett 22, 2021–2024. 11 Fontes R, Dukhovich A, Sillero A & Gu ¨ nther Sillero MA (1997) Synthesis of dehydroluciferin by firefly luci- ferase, Effect of dehydroluciferin, coenzyme A and nucleoside triphosphates on the luminescent reaction. Biochem Biophys Res Comm 237, 445–450. 12 Fontes R, Ortiz B, de Diego A, Sillero A & Gu ¨ nther Sillero MA (1998) Dehydroluciferyl-AMP is the main intermediate in the luciferin dependent synthesis of Ap4A catalyzed by firefly luciferase. FEBS Lett 438, 190–194. 13 Esteves da Silva JCG, Magalha ˜ es JMCS & Fontes R (2001) Identification of enzyme produced firefly oxyluciferin by reverse phase HPLC. Tetrahedron Lett 42, 8172–8176. 14 DeLuca M (1976) Firefly luciferase. Adv Enzymol 44, 37–68. 15 Sillero A & Sillero MA (2000) Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases. Pharmacol Ther 87, 91–102. 16 Nakamura M, Maki S, Amano Y, Ohkita Y, Niwa K, Hirano T, Ohmiya Y & Niwa H (2005) Firefly luciferase exhibits bimodal action depending on the luciferin chirality. Biochem Biophys Res Commun 331, 471–475. 17 Fraga H, Esteves da Silva JCG & Fontes R (2003) pH opposite effects on synthesis of dinucleoside polypho- sphates and on oxidation reactions catalyzed by firefly luciferase. FEBS Lett 543, 37–41. 18 Fraga H, Esteves da Silva JCG & Fontes R (2004) Chemical synthesis and firefly produced dehydroluciferyl coenzyme A. Tetrahedron Lett 45, 2117–2120. 19 Branchini BR, Magyar RA, Murtiashaw MH, Anderson SM & Zimmer M (1998) Site-directed mutagenesis of histidine 245 in firefly luciferase: a proposed model of the active site. Biochemistry 37, 15311–15319. 20 Oba Y, Ojika M & Inouye S (2003) Firefly luciferase is a bifunctional enzyme: ATP-dependent monooxygenase and a long chain fatty acyl-CoA synthetase. FEBS Lett 540, 251–254. 21 Fraga H, Esteves da Silva JCG & Fontes R (2004) Iden- tification of enzyme produced firefly luciferyl-adenylate and the enzyme synthesis of luciferyl-CoA. Chembio- chem 5, 110–115. 22 Hosaka K, Mishina M, Tanaka T, Kamiryo T & Numa S (1979) Acyl-coenzyme A synthetase I from Candida lipolytica. Purification, properties and immunochemical studies. Eur J Biochem 93, 197–203. 23 Tanaka T, Hosaka K, Hoshimaru M & Numa S (1979) Purification and properties of long-chain acyl- coenzyme A synthetase from rat liver. Eur J Biochem 98, 165–172. 24 McElroy WD, DeLuca M & Travis J (1967) Molecular uniformity in biological catalyses. The enzymes con- cerned with firefly luciferin, amino acid, and fatty acid utilization are compared. Science 157, 150–160. 25 Suzuki H, Kawarabayasi Y, Kondo J, Abe T, Nishikawa K, Kimura S, Hashimoto T & Yamamoto T (1990) Structure and regulation of rat long-chain acyl- CoA synthetase. J Biol Chem 265, 8681–8685. 26 Goto T, Kubota I, Suzuki N & Kishi Y (1973) Aspects of the mechanism of bioluminescence. In Chemiluminescence and Bioluminescence (Cormier MJ, Hercules DM & Lee J, eds), pp. 325–335. Plenum Press, New York. 27 Gates BJ & DeLuca M (1975) The production of oxy- luciferin during the firefly luciferase light reaction. Arch Biochem Biophys 169, 616–621. H. Fraga et al. Coenzyme A and luciferase bioluminescence FEBS Journal 272 (2005) 5206–5216 ª 2005 FEBS 5215 [...].. .Coenzyme A and luciferase bioluminescence 28 McElroy WD & Seliger HH (1963) The chemistry of light emission Adv Enzymol 25, 119–166 29 DeLuca M, Wannlund J & McElroy WD (1979) Factors affecting the kinetics of light emission from crude and purified firefly luciferase Anal Biochem 95, 194–198 30 Zako T, Ayabe K, Aburatani T, Kamiya N, Kitayama A, Ueda H & Nagamune T (2003) Luminescent and substrate. .. coenzyme A: a new bioluminescent substract for firefly luciferase in the presence of AMP Angewandte Chemie 117, 3493–3495 34 White EH, McCapra F & Field GF (1963) The structure and synthesis of firefly luciferin J Am Chem Soc 85, 337–343 35 Bowie LJ (1978) Synthesis of firefly luciferin and structural analogs Methods Enzymol 57, 15–28 36 Ames BN (1966) Assay of inorganic phosphate, total phosphate and phosphatases... activities of firefly luciferase N-terminal domain Biochim Biophys Acta 1649, 183–189 31 Lee RT, Denburg JL & McElroy WD (1970) Substratebinding properties of firefly luciferase II ATP-binding site Arch Biochem Biophys 141, 38–52 5216 H Fraga et al 32 Koshland DE Jr (1960) The active site and enzyme action Adv Enzymol 22, 45–97 33 Fraga H, Fontes R & Esteves da Silva JCG (2005) Synthesis of luciferyl coenzyme. .. (1978) Synthesis of firefly luciferin and structural analogs Methods Enzymol 57, 15–28 36 Ames BN (1966) Assay of inorganic phosphate, total phosphate and phosphatases Methods Enzymol 8, 115–118 FEBS Journal 272 (2005) 5206–5216 ª 2005 FEBS . Coenzyme A affects firefly luciferase luminescence because it acts as a substrate and not as an allosteric effector Hugo Fraga 1,2 , Diogo Fernandes 1,2 ,. firefly luciferase. Anal Biochem 95, 194–198. 30 Zako T, Ayabe K, Aburatani T, Kamiya N, Kitayama A, Ueda H & Nagamune T (2003) Luminescent and substrate