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)

´
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: [email protected]
(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
,

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-

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)

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

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-

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.

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

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

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

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

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 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

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

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