Báo cáo Y học: Kinetic and biochemical analyses on the reaction mechanism of a bacterial ATP-citrate lyase ppt

Kinetic and biochemical analyses on the reaction mechanism
of a bacterial ATP-citrate lyase
Tadayoshi Kanao, Toshiaki Fukui, Haruyuki Atomi and Tadayuki Imanaka
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan
The prokaryotic ATP-citrate lyase is considered to be a key
enzyme of the carbon dioxide-fixing reductive tricarboxylic
acid (RTCA) cycle. Kinetic examination of the ATP-citrate
lyase from the green sulfur bacterium Chlorobium limicola
(Cl-ACL), an a
4
b
4
heteromeric enzyme, revealed that the
enzyme displayed typical Michaelis-Menten kinetics toward
ATP with an apparent K
m
value of 0.21 ± 0.04 m
M
.
However, strong negative cooperativity was observed with
respect to citrate binding, with a Hill coefficient (n
H
) of 0.45.
Although the dissociation constant of the first citrate mole-
cule was 0.057 ± 0.008 m
M
, binding of the first citrate
molecule to the enzyme drastically decreased the affinity of
the enzyme for the second molecule by a factor of 23. ADP
was a competitive inhibitor of ATP with a K
i

value of
0.037 ± 0.006 m
M
. Together with previous findings that the
enzyme catalyzed the reaction only in the direction of citrate
cleavage, these kinetic features indicated that Cl-ACL can
regulate both the direction and carbon flux of the RTCA
cycle in C. limicola. Furthermore, in order to gain insight on
the reaction mechanism, we performed biochemical analyses
of Cl-ACL. His273 of the a subunit was indicated to be the
phosphorylated residue in the catalytic center, as both cat-
alytic activity and phosphorylation of the enzyme by ATP
were abolished in an H273A mutant enzyme. We found that
phosphorylation of the subunit was reversible. Nucleotide
preference for activity was in good accordance with the
preference for phosphorylation of the enzyme. Although
residues interacting with nucleotides in the succinyl-CoA
synthetase from Escherichia coli were conserved in AclB,
AclA alone could be phoshorylated with the same nucleotide
specificity observed in the holoenzyme. However, AclB was
necessary for enzyme activity and contributed to enhance
phosphorylation and stabilization of AclA.
Keywords: ATP-citrate lyase; reductive tricarboxylic acid
cycle; Chlorobium limicola.
ATP-citrate lyase (ACL) (EC 4.1.3.8) catalyzes one of the
most complex enzyme reactions in which acetyl-CoA and
oxaloacetate are produced from citrate and CoA with the
hydrolysis of ATP to ADP and phosphate. The enzyme has
received much attention in mammalian cells, as it is
presumed to play a vital role in providing acetyl-CoA and

oxaloacetate in the cytosol as starting materials for a variety
of biosynthetic pathways. Rat and human ACLs from
various organs and tissues have been extensively studied in
terms of biochemical and genetic analyses [1–3], as well as
transcriptional regulation [4] and post-translational phos-
phorylation [5]. Subsequently, ACL has been investigated in
many eukaryotic cells, including fungus [6], yeast [7], and
plant cells [8]. It has been proposed that the eukaryotic ACL
reaction consists of the following three steps:
Enzyme þ Mg
2þ
- ATP ()
Enzyme-PO
2À
3
þ Mg
2þ
-ADP ð1Þ
Enzyme-PO
2À
3
þ citrate () Enzyme-citryl-PO
2À
3
ð2Þ
Enzyme-citryl-PO
2À
3
þ CoA-SH ()
oxaloacetate þ acetyl-CoA+Enzyme ð3Þ

In contrast to the eukaryotic ACLs, little is known about
the prokaryotic ACL. The prokaryotic ACLs have been
identified only from autotrophic bacteria and archaea that
utilize the reductive tricarboxylic acid (RTCA) cycle as a
carbon dioxide (CO
2
) assimilation pathway [9–11]. The
prokaryotic ACLs have been purified and characterized
from a few bacteria [12–14]. Studies with the purified
proteins have raised the possibilities that ACL plays a key
role in controlling the flux in the RTCA cycle.
We have previously isolated the green sulfur bacterium
Chlorobium limicola strain M1, and found that the strain
carries out carbon dioxide fixation via the RTCA cycle. We
have identified and performed initial characterization of
ACL along with NADP-dependent isocitrate dehydroge-
nase from this strain [15,16]. We were able to clone for the
first time the prokaryotic ACL gene, and heterologous gene
expression and characterization of the recombinant protein
revealed that the enzyme (Cl-ACL), unlike its mammalian
counterpart, was comprised of two distinct gene products,
AclA (a subunit) and AclB (b subunit). By comparing the
primary structures of AclA and AclB with that of the
mammalian enzyme, we found that AclA and AclB
Correspondence to T. Imanaka, Department of Synthetic Chemistry
and Biological Chemistry, Graduate School of Engineering, Kyoto
University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606–8501, Japan.
Fax: + 81 75 7534703, Tel.: + 81 75 7535568,
E-mail:
Abbreviations: RTCA cycle, reductive tricarboxylic acid cycle; ACL,

ATP-citrate lyase; Cl-ACL, ATP-citrate lyase from Chlorobium limi-
cola;AclA,a subunit of ATP-citrate lyase; AclB, b subunitofATP-
citrate lyase.
Enzyme: ATP-citrate lyase (EC 4.1.3.8).
(Received 4 February 2002, revised 13 May 2002,
accepted 23 May 2002)
Eur. J. Biochem. 269, 3409–3416 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03016.x
corresponded to the C-terminal (33–39% identical) and
N-terminal (27–34% identical) regions of the single peptide
mammalian ACL, respectively. Cl-ACL did not catalyze the
reverse reaction, the citrate synthase reaction, indicating
that ACL could control the direction of carbon flux in the
RTCA cycle in C. limicola. Furthermore, we found that
Cl-ACL activity was inhibited under the presence of higher
ADP/ATP ratios. This result suggests that the enzyme may
also contribute in regulating the amount of carbon flux in
the cycle depending on the levels of intracellular energy
available from light.
Here, we report a biochemical and kinetic examination of
the bacterial heteromeric ACL from C. limicola,mainly
focusing on the enzyme reaction mechanism. Interesting
kinetic features were observed with the enzyme in terms of
citrate binding, as well as inhibition by ADP. In addition,
our results indicate the steps that govern the nucleotide
dependency of the enzyme and the inhibition observed with
ADP.
MATERIALS AND METHODS
Purification of the recombinant
Cl
-ACL

Construction of the expression vector pETACL harboring
the aclBA genes from C. limicola strain M1, and the
expression procedure of the genes in E. coli BL21(DE3)
have been previously described [15]. The recombinant
enzyme was purified by using A
¨
KTA explorer 10S appar-
atus (Amersham Pharmacia Biotech, Uppsala, Sweden) at
4 °C in all steps. The cell-free extract after ultracentrifuga-
tion (110 000 g) was applied onto HiTrapQ HR5/5 anion
exchange column (Amersham Pharmacia Biotech) equili-
brated with 20 m
M
potassium phosphate buffer (KPB)
(pH 7.4), and the enzyme was eluted with a linear gradient
of KCl (0–0.5
M
). The active fraction was concentrated by
ultrafiltration treatment and was applied onto TSKgel
G4000SW (Tosoh, Tokyo, Japan) gel-filtration column
equilibrated with 20 m
M
KPB containing 0.1
M
KCl. The
active fraction was then applied onto HiTrap Blue HR5/5
affinity column (Amersham Pharmacia Biotech) equilibrat-
ed with 20 m
M
KPB (pH 7.4), and purified ACL was eluted

with a linear gradient of KCl (0–1.0
M
). The homogeneity of
active fractions after each step was confirmed by SDS/
PAGE analysis.
In order to dissociate the AclAB complex, the purified
enzyme was applied on Bio-Scale CHT5-I hydroxyapatite
column (Bio-Rad Laboratories, Hercules, CA, USA) equil-
ibrated with 10 m
M
KPB (pH 7.4). AclB was eluted with a
linear gradient of KPB (10–100 m
M
). After the active
fraction (AclAB complex) was eluted with 100 m
M
KPB
(pH 7.4), AclA was then eluted with 400 m
M
KPB (pH 7.4).
The dissociation of the subunits was confirmed by SDS/
PAGE analysis.
Assay of enzyme activity
ACL activity was assayed by the coupled malate dehydrog-
enase (MDH) method [17]. The reaction mixture contained
10 m
M
MgCl
2
,10m

M
dithiothreitol or 2-mercaptoethanol,
0.2 m
M
NADH, 50 l
M
CoA, 2 m
M
sodium citrate, 1 m
M
ATP, 3.3 U MDH, and recombinant ACL solution in 0.1
M
Tris/HCl buffer (pH 8.4) in a total volume of 1 mL. All
measurements were performed at 30 °C.Oneunitofactivity
wasdefinedas1lmol of NADH oxidized per min. Kinetic
analyses were calculated with SigmaPlot (SPSS Science,
Chicago, IL, USA).
Site-directed mutagenesis
The point mutation was introduced using QuikChange
TM
Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA,
USA). The two complementary oligonucleotide primers
were primer 1; 5¢-GGTATGAAGTTCGGCGCC
GCCGG
TGCCAAGGAAGG-3¢ and primer 2; 5¢-CCTTCCTTGG
CACC
GGCGGCGCCGAACTTCATACC-3¢. The codon
for His273 (CAC) was replaced by an alanine codon (GCC;
underlined). Experiments were carried out as advised by the
manufacturer. Introduction of the point mutation, and

absence of unintended mutations were confirmed by DNA
sequencing. The expression and purification of the mutant
enzyme was performed by the same procedure as that of the
wild-type enzyme as described above.
Phosphorylation of ACL
Phosphorylation of Cl-ACL was measured as follows; Cl-
ACL was incubated with 0.2 lCi [c-
32
P]ATP for 15 min at
30 °C. The 50-lL reaction mixture contained 50 m
M
Tris/
HCl buffer (pH 8.4), 5 m
M
MgCl
2
,and2 m
M
dithiothreitol.
The reaction was terminated by addition of 25 lLSDS/
PAGE loading buffer (3 · concentrated) into the mixture
or by taking a 10-lL aliquot of the mixture and mixing it
with 5 lL loading buffer. SDS/PAGE loading buffer
contained 10% (v/v) 2-mercaptoethanol, 10% (v/v) gly-
cerol, 5% (w/v) SDS, 60 l
M
bromophenol blue (BPB), and
0.1
M
Tris/HCl buffer (pH 6.8). Additional procedures of

these experiments are mentioned in each figure legend. The
samples were applied on SDS/PAGE without heat treat-
ment. After the electrophoresis, the gel was dried by
RAPIDRY gel-dry system (ATTO, Tokyo, Japan) for
90 min at 80 °C and used for autoradiography.
ATP and ADP were separated and detected by thin layer
chromatography (TLC) with a previously described method
[18]. In order to generate [a-
32
P]ADP, [a-
32
P]ATP was
treated with
D
-glucose and hexokinase (Sigma, St Louis,
MO, USA). [a-
32
P]ATP (1 lCi) was added into a reaction
mixture containing 20 m
M
Tris/HCl buffer (pH 7.5),
10 m
MD
-glucose, 10 m
M
MgCl
2
, and 0.5 U of hexokinase.
The reaction mixture was incubated at 30 °Cfor2hand
then treated at 80 °C for 15 min in order to denature

hexokinase. The purified ACL incubated with nonlabeled
ATP at 30 °C for 15 min was mixed together with the
[a-
32
P]ADP, and the reaction mixture was spotted directly
onto Polygram CEL300PEI TLC plates (Macherey-Nagel,
GmbH & Co., Duren, Germany). The substrate and
product of the reaction were separated by one dimension
chromatography using 1
M
LiCl.
RESULTS
Purification of
Cl
-ACL from recombinant
E. coli
Cl-ACL consists of two distinct subunits a (AclA) and b
(AclB), with molecular masses of 65 535 Da and
43 657 Da, respectively. The two subunits were supposed
3410 T. Kanao et al. (Eur. J. Biochem. 269) Ó FEBS 2002
to comprise an (ab)
4
structure, resembling the homotetra-
meric quaternary structure of mammalian ACL. Recom-
binant Cl-ACL was purified from E. coli cells harboring the
aclBA genes from C. limicola strain M1. The purification
procedure was slightly modified from a previous report [15],
and mentioned in Materials and Methods. The homogen-
eity of the recombinant protein was analyzed by SDS/
PAGE (see below).

Kinetic analysis of
Cl
-ACL
Kinetic examination of Cl-ACL with citrate was performed.
The velocity plot and Lineweaver–Burk plot are shown in
Fig. 1A,B, respectively. The 1/v value displayed a steep
decrease at concentrations above 2 m
M
citrate (1/s < 0.5)
(Fig. 1B). This phenomenon suggested negative coopera-
tivity [19] with respect to citrate binding. We calculated the
ratio of substrate concentrations that support V
max
/2 and
V
max
/4, S
0.5
and S
0.25
, respectively. Indeed in the substrate-
velocity plot, S
0.5
/S
0.25
ratio was 9.2, which was well above
the expected ratio of 3 in the case of a nonallosteric enzyme
(Fig. 1A). A Hill plot was constructed, and a slope with an
extremely low value of 0.45 was observed at concentrations
between 0.05 and 2.0 m

M
(Fig. 1C). At concentrations
above 2 m
M
, the slope value was 1.0, indicating that no
allosteric effects were present under these concentrations.
For the calculation of our kinetic results, the velocity data
for citrate were fitted by nonlinear regression analysis with
the following equation [19].
m
V
max
¼
ð½S=K
S
þ 3½S
2
=aK
2
S
þ 3½S
3
=a
2
bK
3
S
þ½S
4
=a

3
b
2
cK
4
S
Þ
ð1 þ 4½S=K
S
þ 6½S
2
=aK
2
S
þ 4½S
3
=a
2
bK
3
S
þ½S
4
=a
3
b
2
cK
4
S

Þ
where v is the initial velocity of the reaction, V
max
is the
maximum velocity, [S] is the concentration of citrate, K
s
is
the dissociation constant of the enzyme-citrate (ES) com-
plex, and a, b, and c are the interaction factors of the first,
second and third substrate molecule(s) toward vacant
substrate binding sites, respectively. In this case, the
dissociation constant of the first citrate molecule K
s1
can
be represented by K
s1
¼ K
s
/4, while K
s2
¼ a2K
s
/3,
K
s3
¼ ab3K
s
/2, and K
s4
¼ abc4K

s
[19]. Our data fitted very
well as represented in Fig. 1A with an R
2
value of 0.999, and
we obtained a V
max
value of 3.7 ± 0.1 U mg
)1
.The
dissociation constants were K
s1
¼ 0.057 ± 0.008 m
M
,
K
s2
¼ 1.3 ± 0.4 m
M
, K
s3
¼ 18 ± 20 m
M
,andK
s4
¼
1.6 ± 2.0 m
M
. These facts clearly indicated that Cl-ACL
was an allosteric enzyme displaying strong negative coop-

erativity in citrate binding. Consequently, the S
0.5
was high
for citrate, with a value of 2.5 m
M
.
InthecaseofATP,Cl-ACL exhibited typical Michaelis-
Menten kinetics with an apparent Michaelis constant (K
m
value) of 0.21 ± 0.04 m
M
(Fig. 2A). We previously found
the activity of Cl-ACL was strongly inhibited with a higher
ratio of ADP to ATP [15], with 50% inhibition observed at
an equimolar ratio. Lineweaver-Burk plots with or without
ADP against ATP are shown in Fig. 2B. The plots indicated
that ADP was a competitive inhibitor of ATP. The
inhibition constant, or K
i
value, for ADP was determined
to be 0.037 ± 0.006 m
M
.
Nucleotide dependency
The effect of different nucleotides (ATP, GTP, CTP, UTP,
and dATP) on ACL activity was re-examined by using a
malate dehydrogenase (MDH)-linked assay, a much more
sensitive and accurate assay than the hydroxamate method
used in our previous report [15]. Each nucleotide was
supplied at a concentration of 1 m

M
into the reaction
mixture. The results are shown in Table 1. The enzyme
exhibited maximum activity in the presence of ATP, and in
the presence of dATP showed 40% of this activity. In the
presence of CTP, activity was slightly observed (less than
0.2%), whereas no activity could be detected with GTP and
UTP. This nucleotide dependency showed the same ten-
dency as the previous result, although the relative activities
with CTP and dATP were determined to be lower.
A single mutation H273A in AclA
In the human ACL, the His765 residue has been identified
as the catalytic site which is autophosphorylated by ATP-
Mg
2+
in the first step of the reaction [3]. It was also
suggested that phosphohistidine was generated by ATP-
Mg
2+
in the enzyme from the green sulfur bacterium
C. tepidum [14]. Comparison of primary structure predicted
Fig. 1. Kinetic analysis of recombinant Cl-ACL with citrate. (A) Velocity plot with various concentrations of citrate. The solid curve was fitted by
nonlinear regression analysis of the experimental data as described in the text. The dotted line represents a calculated curve of activity if the enzyme
were to follow typical Michaelis–Menten kinetics. The small panel is focused on lower citrate concentrations (0–4 m
M
). (B) A Lineweaver–Burk
plot of the kinetic data. (C) A Hill plot of the kinetic data. Hill coefficients are indicated as n
H
values. The n
H

value of 0.45 is given in the lower citrate
concentrations (0.05–2 m
M
).
Ó FEBS 2002 ATP-citrate lyase from Chlorobium limicola (Eur. J. Biochem. 269) 3411
His273 on AclA as the phosphorylated catalytic site. A
mutant gene was constructed in which His273 residue was
replaced with alanine, and subsequently coexpressed in
E. coli BL21(DE3) cells together with aclB. The H273A
mutant protein was purified as a heteromeric enzyme with
identical elution profiles to those of the wild type ACL in the
purification steps (data not shown). The results indicate that
the mutation did not affect the subunit assembly of the
enzyme. However, no ACL activity was observed in the
mutant protein, indicating that His273 played an essential
role in the activity of the enzyme, most likely as the residue
phosphorylated by ATP.
Phosphorylation of
Cl
-ACL
In order to investigate the reaction mechanism of prokary-
otic ACL, we examined whether the enzyme was phos-
phorylated with the c-phosphate group of ATP. When
incubated with [c-
32
P]ATP, the 65 kDa subunit corres-
ponding to AclA was phosphorylated, but the 40 kDa
subunit (AclB) was not (Fig. 3A, lane 1). As substrates
other than ATP were not added in the reaction mixture, this
indicated that phosphorylation of the protein can occur

prior to the interactions with other substrates such as citrate
or CoA. Furthermore, the H273A mutant enzyme was not
phosphorylated under these conditions (Fig. 3A, lane 2),
indicating that His273 in AclA is phosphorylated by ATP,
and that this is essential for enzyme activity. Moreover, we
have found that the phosphorylated protein was dephos-
phorylated in the presence of citrate (Fig. 3A, lane 4).
Examination with thin layer chromatography also displayed
the specific release of the phosphate group with the addition
of citrate, suggesting the transfer of the labeled phosphate
from the enzyme to citrate (data not shown). The results
support an ordered mechanism of the enzyme reaction
where the phosphate group of ATP is first covalently bound
to the His273 catalytic residue, and subsequently transferred
to the citrate molecule to form citryl phosphate.
Enzyme activity of Cl-ACL displayed a high preference
for ATP (100%), followed by dATP (40%) and CTP
(0.2%). Taking into account the results of the above
experiments, this preference can be assumed to reflect the
phosphorylation efficiencies by various nucleotides. We
have clarified this by examining the phosphorylation of the
AclA with various [c-
32
P]NTPs. Results with c-
32
P-labeled
ATP (0.2 lCi), GTP (5 lCi), CTP (5 lCi), and dATP
(0.5 lCi) are shown in Fig. 4. Radioactive signals corres-
ponding to phosphorylated AclA were observed in the
reaction mixtures when ATP, CTP, and dATP were used as

a phosphate donor. The intensities of the signals were in
good accordance with the activity levels observed for each
nucleotide. Efficient dephosphorylation of the phosphoryl-
ated proteins was observed upon addition of citrate,
regardless of the nucleotide that provided the phosphate
group.
Subunit dissociation of
Cl
-ACL
We have previously described that the two subunits of
Cl-ACL (AclA and AclB) could be dissociated from each
other by hydroxyapatite column chromatography [15]. In
order to clarify the role of each subunit, AclA and AclB
were dissociated and subjected to further individual exam-
ination. The homogeneity of the purified wild type and
H273A mutant ACL were examined by SDS/PAGE
(Fig. 5A, lanes 1 and 2). Efficient dissociation of the
individual subunits of the wild type ACL was confirmed in
lanes 3 and 4. The individual subunits of the H273A mutant
Fig. 2. Kinetic analysis of recombinant
Cl-ACL with ATP and its inhibition by ADP.
(A) Lineweaver–Burk plots for various con-
centrations of ATP. (B) Double reciprocal
plots for various concentrations of ATP with
or without ADP. ADP concentrations were
0m
M
(circles), 0.1 m
M
(squares), and 0.3 m

M
(triangles).
Table 1. ACL activities for each nucleotide. NA, no activity.
Nucleotides (1 m
M
) ATP dATP CTP GTP UTP
Activity (UÆmg
)1
) 1.30 0.52 Trace NA NA
Activity (%) 100 40 < 0.2 0 0
Fig. 3. Phosphorylation and dephosphorylation of Cl-ACL and the
H273A mutant protein. Samples were subjected to SDS/PAGE
(12.5%) and autoradiography. Lane 1, purified wild type enzyme was
incubated with [c-
32
P]ATP (0.2 lCi) for 15 min at 30 °C; lane 2,
purified H273A mutant protein incubated with [c-
32
P]ATP (0.2 lCi)
for 15 min at 30 °C; lane 3, same as in lane 1 before addition of 2 m
M
citrate; lane 4, enzyme after addition of 2 m
M
citrate and incubation
for 15 min at 30 °C with the sample in lane 3. Molecular masses (kDa)
are indicated on the left of the panel.
3412 T. Kanao et al. (Eur. J. Biochem. 269) Ó FEBS 2002
ACL were also observed in lanes 5 and 6. No activity could
be detected in the fractions containing individual subunits
(Fig. 5A, lane 3 and 4). Interestingly, up to 70% of activity

was recovered when the individual subunit fractions (AclA
and AclB) were mixed together and incubated at 25 °Cfor
5min.
Phosphorylation of AclA with or without AclB
The catalytic residue that is the target for phosphorylation
(His273) is located on the AclA subunit, whereas a sequence
comparison with bacterial succinyl-CoA synthetase dis-
played that residues interacting with ATP were conserved in
the AclB subunit (K50, R58, E97, E104, N190, and D205)
[15]. The holoenzyme and individual subunits of both wild
type and H273A mutant ACL were incubated with
[c-
32
P]ATP. Although no ACL activity was detected in
the AclA fraction without AclB, we found that in the case of
the wild type enzyme, AclA alone was phosphorylated by
[c-
32
P]ATP to a similar extent as the AclAB complex after
90 min (Fig. 5B, lane 2). We then presumed that AclB
might be responsible for nucleotide specificity, and not for
phosphorylation itself. However, further examinations
proved otherwise. No significant difference was observed
in the nucleotide specificity of AclA phosphorylation
between the AclAB complex (holoenzyme) and the AclA
subunit alone (Fig. 6). We then followed the phosphoryla-
tion levels of the AclAB complex and AclA subunit at
various time intervals. The phosphorylation by [c-
32
P]ATP

was performed at 30 °C, and aliquot samples after desired
periods were applied to SDS/PAGE. Although a prompt
phosphorylation was observed in the holoenzyme (Fig. 7A),
the AclA fraction was phosphorylated at a much slower rate
(Fig. 7B). Furthermore, a labeled degradation product of
the AclA subunit was observed in the absence of the AclB
subunit (Figs 7B and 5B). However, the actual amount of
the degradation product was very low, as it could not be
detected in Coomassie Brilliant Blue (CBB) stained gels
before (Fig. 5A, lanes 3 and 5) or after (data not shown)
incubation with ATP. The relatively high intensity of the
degradation product in the autoradiograph compared to
the CBB stained gels indicated that a high percentage of the
degradation product was phosphorylated. The phosphory-
lation of the AclA protein may lead to a slight decrease in
stability of the protein. As no signals were detected in the
case of the H273A mutant protein (Fig. 5B, lanes 4 and 5),
the phosphorylation of the AclA protein and its degradation
product is most likely to occur specifically at the His273
Fig. 4. Phosphorylation of Cl-ACL with various [c-
32
P]NTPs. The
nucleotides which were used in each reaction are indicated above the
lanes. The enzyme was incubated with the indicated nucleotide at
30 °C for 15 min. Samples were subjected to SDS/PAGE (12.5%) and
autoradiography. The mixtures before addition of 2 m
M
citrate are
indicated with (–), while those after addition of 2 m
M

citrate are
indicated with (+). Nucleotides were added at concentrations of
0.2 lCi for [c-
32
P]ATP, 5 lCi for [c-
32
P]GTP, 5 lCi for [c-
32
P]CTP,
and 0.5 lCi for [c-
32
P]dATP. Molecular masses (kDa) are indicated on
theleftofthepanel.
Fig. 5. Dissociation of AclAB. (A) SDS/PAGE of wild type and
H273A mutant enzymes and their individual subunits. Samples were
applied to a 12.5% gel, and then stained with Coomassie Brilliant Blue
R250. Purified wild type and H273A mutant enzymes were applied
onto lanes 1 and 2, respectively. The subunits AclA and AclB of wild
type and H273A Cl-ACL were dissociated by hydroxyapatite column
chromatography. AclB eluted with 10–100 m
M
KPB (lanes 4 and 6),
while AclA eluted with 400 m
M
KPB (lanes 3 and 5). The dissociated
subunits are derived from the enzymes which are indicated above the
lanes (WT; wild-type Cl-ACL, H273A; H273A mutant protein). Lane
M, molecular marker. (B) Autoradiograph of wild type (lanes 1–3) and
H273A mutant (lanes 4–6) enzymes and their individual subunits after
incubation with 0.2 lCi of [c-

32
P]ATP for 90 min. Each reaction
mixture contained 1 lg protein. Purified enzymes before subunit dis-
sociation (AB) are shown in lanes 1 and 4, the individual AclA subunits
(a) are shown in lanes 2 and 5, and AclB subunits (b) in lanes 3 and 6.
Molecular masses (kDa) are indicated on the side of each panel. The
asterisk indicates the degradation product of AclA described in the
text.
Fig. 6. Nucleotide specificities of AclA phosphorylation with or without
AclB. Nucleotides used for each reaction are indicated above the lanes.
Each reaction mixture contained 1 lg protein and was incubated with
the indicated nucleotide at 30 °C for 15 min. Samples were subjected
to SDS/PAGE (12.5%) and autoradiography. Nucleotides were added
at concentrations of 0.2 lCi for [c-
32
P]ATP, 5 lCi for [c-
32
P]GTP,
5 lCi for [c-
32
P]CTP, and 0.5 lCi for [c-
32
P]dATP. Molecular masses
(kDa) are indicated on the right of the panel. Samples using purified
enzyme before subunit dissociation and those with the AclA subunit
alone are indicated with ÔABÕ and ÔaÕ, respectively. The asterisk indi-
cates the degradation product of AclA described in the text.
Ó FEBS 2002 ATP-citrate lyase from Chlorobium limicola (Eur. J. Biochem. 269) 3413
residue. Our results suggest that the AclB subunit contri-
butes in enhancing the efficiency of phosphorylation of the

AclA subunit, as well as stabilizing its structure.
Examination of the inhibitory effect of ADP
As an increased ratio of ADP towards ATP significantly
inhibits Cl-ACL activity, we investigated the effect of ADP
on the phosphorylation of AclA. Addition of 10–100 l
M
ADP to the phosphorylated Cl-ACL resulted in dephos-
phorylation of the enzyme (Fig. 8A, lanes 2–5). This
tendency increased with higher concentrations of ADP
(data not shown). An apparent inhibitory effect on AclA
phosphorylation was observed with an increase in ADP
concentration (Fig. 8A, lanes 6–10), indicating a competi-
tion of the labeled phosphate group between enzyme and
ADP. In order to elucidate the fate of the phosphate group
after the enzyme is dephosphorylated by ADP, the follow-
ing experiment was carried out. [a-
32
P]ADP was produced
by treating [a-
32
P]ATP with hexokinase and glucose
(Fig. 8B, lane 2). Purified enzyme was phosphorylated
using unlabeled ATP. The phosphorylated enzyme and
[a-
32
P]ADP were incubated together, and the reaction
product was applied to TLC (Fig. 8B, lane 3). We clearly
detected the generation of ATP in the lane. These results
leave no doubt that the first step of the ACL reaction,
phosphorylation by ATP, is reversible.

DISCUSSION
We have examined the kinetic and biochemical features of a
prokaryotic ATP-citrate lyase. The results provide the first
insight on the reaction mechanism of an ATP-citrate lyase
with a heteromeric structure, and display some interesting
features of the enzyme.
The His273 residue, located on AclA, is phosphorylated
by c-phosphate of ATP. This phosphorylation can take
place in the absence of other substrates, and was found to be
reversible. These results coincided with the previously
reportedfeaturesofmammalianACL[20]andsuccinyl-
CoA synthetase from E. coli (Ec-SCS) displaying similarit-
ies to ACLs in terms of structure and catalytic mechanism
[21,22]. In addition to these results, we demonstrated that
the AclA subunit alone could be phosphorylated when
incubated with ATP, and AclB markedly accelerated the
phosphorylation rate of AclA. Similar results were also
reported in the case of Ec-SCS [21], although the phos-
phorylation of the a-subunit alone (80% of the subunit after
24 h) was much slower than AclA alone (90 min). A major
contribution of AclB, as well as the b subunit of Ec-SCS, in
the catalytic process seems to be assistance of the nucleotide
binding to and phosphorylation of the a subunit. Another
function of AclB was found to be stabilization of the
enzyme, as AclB prevented the degradation of AclA that
was otherwise observed in the absence of AclB.
After the phosphorylation, we detected that the addition
of citrate subsequently removed the phosphate from the
enzyme, presumably forming citryl phosphate. This
occurred in the absence of CoA. Taking into account the

reaction mechanism of mammalian ACL [23], the final step
of the reaction can be assumed to be the nucleophilic attack
of CoA to the phosphorylated carbonyl carbon of citryl
phosphate, and the cleavage of the resulting citryl-CoA to
acetyl-CoA and oxaloacetate. In our previous report, we
could not detect citrate synthase activity in Cl-ACL [15]. It
has been described that the reaction of mammalian ACL
was reversible, although it is much stronger in the cleavage
direction [20]. Since the phosphorylation of ACLs was
reversible, the unidirectional characteristics of the enzymes
were likely to be due to the low efficiencies in the
condensation between acetyl-CoA and oxaloacetate.
We also revealed that the nucleotide specificity of the
phosphorylation of AclA alone displayed the same tendency
with the overall enzyme reaction. This finding suggests that
the nucleotide is discriminated by AclA, and the phos-
phorylation step governs the overall nucleotide specificity of
the holoenzyme. It has been reported that a histidine residue
in ACL from rat liver was autophosphorylated by GTP
even though GTP did not support the overall reaction [24].
This was not the case in Cl-ACL, as GTP did not lead to
phosphorylation of the enzyme, nor support activity.
Fig. 7. Effect of AclB on the phosphorylation of AclA. AclA with (A) or
without AclB (B) was incubated with 0.2 lCi of [c-
32
P]ATP at 30 °C.
The incubation times (min) are indicated above each lane. Samples
were subjected to SDS/PAGE (12.5%) and autoradiography. Equal
amounts of AclA (0.11 pmol) were used in (A) (purified enzyme before
subunit dissociation) and (B) (AclA alone). Molecular masses (kDa)

are indicated on the left of the panel. The asterisk indicates the deg-
radation product of AclA described in the text.
Fig. 8. Effects of ADP on the phosphorylation of Cl-ACL by ATP. (A)
After the following reactions, each sample was subjected to SDS/
PAGE (12.5%) and autoradiography. Purified Cl-ACL after incuba-
tion with 0.2 lCi of [c-
32
P]ATP at 30 °C for 15 min (lane 1). Samples
of lane 1 with addition of 100 l
M
(lane 2), 50 l
M
(lane 3), 20 l
M
(lane
4) and 10 l
M
(lane 5) ADP were further incubated at 30 °Cfor15 min.
Purified Cl-ACL was incubated with 0.2 lCi of [c-
32
P]ATP in the
presence of 100 l
M
(lane 6), 50 l
M
(lane 7), 20 l
M
(lane 8), 10 l
M
(lane

9) and absence (lane 10) of ADP at 30 °C for 15 min. (B) Autoradi-
ograph of TLC. Lane 1, [a-
32
P]ATP; lane 2, [a-
32
P]ATP treated with
hexokinase and glucose; lane 3; sample from lane 2 after incubation
with phosphorylated Cl-ACL (AB-p) for 15 min at 30 °C.
3414 T. Kanao et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Although the nucleotide preference in the phosphorylation
of Ec-SCS has not been examined, a recent crystal structure
of Ec-SCS revealed that the residues interacting with
nucleotides were mostly located in the b-subunit [25]. In
addition, ATP- and GTP-specific SCS isozymes from
pigeon breast and liver are composed of the same asubunit
but different b subunits, indicating the importance of the
b subunit in the nucleotide preference [26]. As alignment of
Cl-ACL with Ec-SCS identified the corresponding residues
for the interaction to nucleotides in AclB, AclB may also
participate in the binding and recognition of nucleotides.
With respect to Cl-ACL, some nucleotide binding residues,
sufficient for nucleotide discrimination, should at least be
present in AclA.
In our kinetic studies, the most striking feature of Cl-ACL
was the strong negative cooperativity observed towards
citrate binding. As K
s1
<<K
s2
<<K

s3
, binding of the first
and second molecules of citrate drastically decrease the
affinity of the enzyme for the second and third molecules,
respectively. The dissociation constant of the first citrate
molecule (K
s1
) was 0.057 ± 0.008 m
M
, lower than the K
m
values of ACLs from other sources [14]. However, the K
s2
value was 23-fold higher, with a value of 1.3 ± 0.4 m
M
,and
the K
s3
value was even higher at 18 ± 20 m
M
. Owing to this
strong negative cooperativity, it can be presumed that under
physiological conditions, the majority of Cl-ACL binds to
only one molecule of citrate. In the presence of sufficient
levels of ATP, the low K
s1
value would contribute to the
efficient conversion of citrate at low concentrations. If
citrate were to accumulate in the cells, the high K
s2

and K
s3
values due to strong negative cooperativity would limit the
reaction rate in comparison with a nonallosteric enzyme.
This feature of Cl-ACL would serve as a valve that limits the
flux of the RTCA cycle in C. limicola.
In the literature, we found that while the ACL from
C. tepidum displayed typical Michaelis–Menten kinetics
[14], the unphosphorylated human ACL showed similar
negative cooperativity toward citrate [5]. The Hill coefficient
was 0.65, indicating a weaker negative cooperativity than
that of Cl-ACL. This negative cooperativity was abolished
when the enzyme was phosphorylated either by cAMP-
dependent protein kinase alone or in combination with
glycogen synthase kinase-3 [27–30]. These kinases phos-
phorylate the Thr446, Ser450, and Ser454 residues of
human ACL. As corresponding residues are not present in
Cl-ACL [15], it is likely that this sort of absolving
mechanism does not exist in Cl-ACL.
Another feature of Cl-ACL was the inhibition observed
with ADP. It has been reported that ADP inhibited the
activities of both mammalian and bacterial ACLs [12,13,31].
The double reciprocal plots with or without ADP showed
that ADP was a competitive inhibitor of ATP with a K
i
value of 0.037 ± 0.006 m
M
. This would result in a decrease
in Cl-ACL activity when intracellular energy is at an
insufficient level. Together with the negative cooperativity

and the unidirectional features of the enzyme, Cl-ACL is
likely to regulate both the direction and carbon flux of the
RTCA cycle in C. limicola.
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