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

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

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

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

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

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).

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

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

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

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

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

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

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-

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.

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)

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

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

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