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:
(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
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