At least part of the light decay is due to the luciferase catalysed formation of dehydroadenylate L-AMP, a by-product that results from oxidation of luciferyl-adenylate LH2-AMP, and is a
Trang 1because it acts as a substrate and not as an allosteric
effector
Hugo Fraga1,2, Diogo Fernandes1,2, Rui Fontes2and Joaquim C G Esteves da Silva1
1 Departmento de Quı´mica, Faculdade de Cieˆncias 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 (LH2), in the
pres-ence of ATP and Mg2+, giving rise to light [1,2] The
bioluminescence reaction involves the reaction of LH2
and ATP to form luciferyl-adenylate (LH2-AMP)
(Reaction 1 and Fig 1) LH2-AMP is then oxidized
by molecular oxygen and, through a series of
inter-mediates, gives rise to AMP, inorganic pyrophosphate
(PPi), CO2 and oxyluciferin (Reaction 2 and Fig 1), the presumed light emitter [2]
LUCþ LH2þ ATP Ð LUCÆLH2-AMPþ PPi ð1Þ LUCÆLH2-AMPþ O2! LUC þ AMP þ CO2
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 Cieˆncias 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 dehydroadenylate (L-AMP), a by-product that results from oxidation of luciferyl-adenylate (LH2-AMP), and is a powerful inhibitor of the bioluminescence reaction (IC50¼ 6 nm) 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 (IC50¼ 5 lm) Moreover, the Vmax for L-CoA formation was determined as 160 min)1, which is one order of magnitude higher than the Vmax 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 LH2-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;
LH2-AMP, luciferyl-adenylate; LH2-CoA, luciferyl-coenzyme A; PPase, inorganic pyrophosphatase; PPi, inorganic pyrophosphate; RLU, relative light units.
Trang 2because of its high sensitivity and specificity [3]
Com-mercial ATP assay kits, apart from LH2 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 LH2-AMP and L results from the
pyrophos-phorolysis of L-AMP (Reaction 3 and Fig 1)
LUCÆL-AMPþ 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-AMPþ 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 LH2 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 LH2 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).
Trang 3LUC, the decay without CoA and the stabilizing effect
of CoA were more pronounced when high
concentra-tions of ATP and LH2 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
LH2-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 LH2and
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 n M ) supplemented (solid symbols) or not supplemented (open symbols) with CoA (50 l M ) 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 l M ) 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.
Trang 4the 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 incubareac-tion of
30 lm L-AMP with various concentrations of CoA and LUC allowed us to estimate the Vmax for L-CoA formation as 160 min)1 This velocity is one order of magnitude higher than the Vmax 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 LH2 transformation into L-AMP and
non-L products in the first 15 s of reaction was 2 min)1 As the Vmaxvalues 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 Vmax 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).
Trang 5Study 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 supplemeninjec-ted 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-acetyl-CoA was incuba-ted with L-AMP in the presence of LUC, we detecincuba-ted 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, MgCl2, LH2and ATP and different concentrations of L-AMP (zero in A, 0.5 l M in B and 10 l M in C) After 60 s, 50 lL of a solution (100 l M ) of CoA (diamonds; uppermost curve), dephospho-CoA (squares), dethio-CoA (circles) or water (continuous line) was injected All the indicated quantities are final concentrations.
Trang 6we 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 Kmof CoA in the thiolytic reaction
and the apparent Ka 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 LH2and ATP The degree of activation has
been defined in Fig 4.
Trang 7contamination of commercial preparations of
acetyl-CoA with acetyl-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, MgCl2,
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 (Ki¼ 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 LH2(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 LH2 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 n M ) 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.
Trang 8produced 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 LH2 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 LH2 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
LH2 (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
LH2or 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-LH2-AMP
and CoA, giving rise to luciferyl-CoA (LH2-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 LH2-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 LH2 and ATP are used, the fraction of LH2-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 lm 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 LH2, 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
Trang 9obtain LH2-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 (24–27C) 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 lL of a
mixture of ATP (50 lm) and LH2(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), MgCl2 (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 MgCl2, 300 lm
acetic acid, 1.5 mm commercial acetyl-CoA, PPase (2 lg of
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 MgCl2, Hepes pH 8.2, L-AMP
and LUC The light reaction was initiated by injecting 50 lL
of a mixture containing 20 lm LH2and 300 lm ATP After
the injection the concentrations of MgCl2(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), MgCl2
(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 MgCl2was incubated for 5 min and then treated for 15 min with alkaline phosphatase (1.2 lg of 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 lm, respectively) into assay tubes that contained Hepes pH 8.2, MgCl2and 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 lm L-AMP, 50 mm Hepes pH 8.2,
2 mm MgCl2, 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 incubaaddi-tion, 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
1 mLÆmin)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 LH2(10 lm) and ATP (50 lm) were coinjected with CoA or dephospho-CoA
Acknowledgements
Financial support from Fundac¸a˜o para a Cieˆncia e Tecnologia (Lisboa) (FSE-FEDER) (Project POCTI⁄ QUI⁄ 37768 ⁄ 2001) (PhD grant SFRH ⁄ BD ⁄ 1395 to Hugo Fraga) is acknowledged We also acknowledge Programa Cieˆncia Viva (Diogo Fernandes) and Abel Duarte (Instituto Superior de Engenharia do Porto) for his help in the construction of the luminometer
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