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

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

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 a4b4heteromeric enzyme, revealed that the

enzyme displayed typical Michaelis-Menten kinetics toward

ATP with an apparent Km value of 0.21 ± 0.04 mM

However, strong negative cooperativity was observed with

respect to citrate binding, with a Hill coefficient (nH)of 0.45

Although the dissociation constant of the first citrate

mole-cule was 0.057 ± 0.008 mM, 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 Ki value of

0.037 ± 0.006 mM 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:

Enzymeþ Mg2þ- ATP()

Enzyme-PO23 þ citrate () Enzyme-citryl-PO23 ð2Þ Enzyme-citryl-PO23 þ CoA-SH ()

oxaloacetateþ 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 (CO2)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: imanaka@sbchem.kyoto-u.ac.jp

Abbreviations: RTCA cycle, reductive tricarboxylic acid cycle; ACL,

ATP-citrate lyase; Cl-ACL, ATP-citrate lyase from Chlorobium

limi-cola; AclA, a subunit of citrate lyase; AclB, b subunit of

ATP-citrate lyase.

Enzyme: ATP-citrate lyase (EC 4.1.3.8).

(Received 4 February 2002, revised 13 May 2002,

accepted 23 May 2002)

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

M A T E R I A L S A N D M E T H O D S

Purification of the recombinantCl -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

4C 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 mM potassium phosphate buffer (KPB)

(pH 7.4), and the enzyme was eluted with a linear gradient

of KCl (0–0.5M) The active fraction was concentrated by

ultrafiltration treatment and was applied onto TSKgel

G4000SW (Tosoh, Tokyo, Japan)gel-filtration column

equilibrated with 20 mMKPB containing 0.1M KCl The

active fraction was then applied onto HiTrap Blue HR5/5

affinity column (Amersham Pharmacia

Biotech)equilibrat-ed with 20 mMKPB (pH 7.4), and purified ACL was eluted

with a linear gradient of KCl (0–1.0M) 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 mMKPB (pH 7.4) AclB was eluted with a

linear gradient of KPB (10–100 mM) After the active

fraction (AclAB complex)was eluted with 100 mM KPB

(pH 7.4), AclA was then eluted with 400 mMKPB (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 mMMgCl2, 10 mMdithiothreitol or 2-mercaptoethanol,

0.2 mMNADH, 50 lMCoA, 2 mMsodium citrate, 1 mM

ATP, 3.3 U MDH, and recombinant ACL solution in 0.1M

Tris/HCl buffer (pH 8.4)in a total volume of 1 mL All

measurements were performed at 30C One unit of activity was defined as 1 lmol 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 QuikChangeTM Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) The two complementary oligonucleotide primers were primer 1; 5¢-GGTATGAAGTTCGGCGCCGCCGG TGCCAAGGAAGG-3¢ and primer 2; 5¢-CCTTCCTTGG CACCGGCGGCGCCGAACTTCATACC-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 ACL was measured as follows; Cl-ACL was incubated with 0.2 lCi [c-32P]ATP for 15 min at

30C The 50-lL reaction mixture contained 50 mMTris/ HCl buffer (pH 8.4), 5 mMMgCl2, and 2 mMdithiothreitol The reaction was terminated by addition of 25 lL SDS/ 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 lMbromophenol blue (BPB), and 0.1M 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 80C 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-32P]ADP, [a-32P]ATP was treated withD-glucose and hexokinase (Sigma, St Louis,

MO, USA) [a-32P]ATP (1 lCi)was added into a reaction mixture containing 20 mM Tris/HCl buffer (pH 7.5),

10 mM D-glucose, 10 mMMgCl2, and 0.5 U of hexokinase The reaction mixture was incubated at 30C for 2 h and then treated at 80C for 15 min in order to denature hexokinase The purified ACL incubated with nonlabeled ATP at 30C for 15 min was mixed together with the [a-32P]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 1MLiCl

R E S U L T S

Purification ofCl-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

to comprise an (ab)4structure, resembling the

homotetra-meric quaternary structure of mammalian ACL

Recom-binant Cl-ACL was purified from E coli cells harboring the

aclBAgenes 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 ofCl-ACL

Kinetic examination of Cl-ACL with citrate was performed

The velocity plot and LineweaverỜBurk plot are shown in

Fig 1A,B, respectively The 1/v value displayed a steep

decrease at concentrations above 2 mMcitrate (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 Vmax/2 and

Vmax/4, S0.5and S0.25, respectively Indeed in the

substrate-velocity plot, S0.5/S0.25ratio 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 mM (Fig 1C) At concentrations

above 2 mM, 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

VmaxỬ

đơS =KSợ 3ơS 2=aK2Sợ 3ơS 3= 2bK3Sợ ơS 4= 3b2cK4Sỡ

đ1 ợ 4ơS =KSợ 6ơS 2=aK2Sợ 4ơS 3= 2bK3Sợ ơS 4= 3b2cK4Sỡ

where v is the initial velocity of the reaction, Vmaxis the

maximum velocity, [S] is the concentration of citrate, Ksis

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 Ks1 can

be represented by Ks1Ử Ks/4, while Ks2Ử a2Ks/3,

Ks3Ử ab3Ks/2, and Ks4Ử abc4Ks[19] Our data fitted very

well as represented in Fig 1A with an R2value of 0.999, and

we obtained a Vmax value of 3.7 ổ 0.1 U mg)1 The dissociation constants were Ks1Ử 0.057 ổ 0.008 mM,

Ks2Ử 1.3 ổ 0.4 mM, Ks3Ử 18 ổ 20 mM, and Ks4Ử 1.6 ổ 2.0 mM These facts clearly indicated that Cl-ACL was an allosteric enzyme displaying strong negative coop-erativity in citrate binding Consequently, the S0.5was high for citrate, with a value of 2.5 mM

In the case of ATP, Cl-ACL exhibited typical Michaelis-Menten kinetics with an apparent Michaelis constant (Km value)of 0.21 ổ 0.04 mM(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 Kivalue, for ADP was determined

to be 0.037 ổ 0.006 mM 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 mM 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-Mg2+ in the first step of the reaction [3] It was also suggested that phosphohistidine was generated by

ATP-Mg2+ 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 MichaelisỜMenten kinetics The small panel is focused on lower citrate concentrations (0Ờ4 m M ) (B) A LineweaverỜBurk 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 (0.05Ờ2 m ).

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 ofCl -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-32P]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-32P]NTPs Results with c-32P-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 ofCl -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

0 m 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

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-32P]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.

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 25C for

5 min

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-32P]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-32P]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-32P]ATP was performed at 30C, 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-32P]ATP, 5 lCi for [c-32P]GTP, 5 lCi for [c-32P]CTP,

and 0.5 lCi for [c- 32 P]dATP Molecular masses (kDa)are indicated on

the left of the panel.

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 AB and a, respectively The asterisk indi-cates the degradation product of AclA described in the text.

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 lM

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-32P]ADP was produced

by treating [a-32P]ATP with hexokinase and glucose (Fig 8B, lane 2) Purified enzyme was phosphorylated using unlabeled ATP The phosphorylated enzyme and [a-32P]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

D I S C U S S I O N

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 reported features of mammalian ACL [20] and succinyl-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-32P]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-32P]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 C for 15 min.

Purified Cl-ACL was incubated with 0.2 lCi of [c-32P]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-32P]ATP; lane 2, [a-32P]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.

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 a subunit

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 Ks1<<Ks2<<Ks3, 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 (Ks1)was 0.057 ± 0.008 mM, lower than the Km

values of ACLs from other sources [14] However, the Ks2

value was 23-fold higher, with a value of 1.3 ± 0.4 mM, and

the Ks3value was even higher at 18 ± 20 mM 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 Ks1value would contribute to the

efficient conversion of citrate at low concentrations If

citrate were to accumulate in the cells, the high Ks2and Ks3

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 Ki

value of 0.037 ± 0.006 mM 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

R E F E R E N C E S

1 Houston, B & Nimmo, H.G (1984)Purification and some kinetic

properties of rat liver ATP citrate lyase Biochem J 224, 437–443.

2 Elshourbagy, N.A., Near, J.C., Kmetz, P.J., Sathe, G.M., Southan, C., Strickler, J.E., Gross, M., Young, J.F., Wells, T.N.C.

& Groot, P.H.E (1990)Rat ATP citrate-lyase Molecular cloning and sequence analysis of a full-length cDNA and mRNA abun-dance as a function of diet, organ, and age J Biol Chem 265, 1430–1435.

3 Elshourbagy, N.A., Near, J.C., Kmetz, P.J., Wells, T.N.C., Groot, P.H.E., Saxty, B.A., Hughes, S.A., Franklin, M & Gloger, I.S (1992)Cloning and expression of a human ATP-citrate lyase cDNA Eur J Biochem 204, 491–499.

4 Sato, R., Okamoto, A., Inoue, J., Miyamoto, W., Sakai, Y., Emoto, N., Shimano, H & Maeda, M (2000)Transcriptional regulation of the ATP citrate-lyase gene by sterol regulatory ele-ment-binding proteins J Biol Chem 275, 12497–12502.

5 Potapova, I.A., El-Maghrabi, M.R., Doronin, S.V & Benjamin, W.B (2000)Phosphorylation of recombinant human ATP: citrate lyase by cAMP- dependent protein kinase abolishes homotropic allosteric regulation of the enzyme by citrate and increases the enzyme activity Allosteric activation of ATP: citrate lyase by phosphorylated sugars Biochemistry 39, 1169–1179.

6 Pfitzner, A., Kubicek, C.P & Ro¨hr, M (1987)Presence and reg-ulation of ATP: citrate lyase from the citric acid producing fungus Aspergillus niger Arch Microbiol 147, 88–91.

7 Shashi, K., Bachhawat, A.K & Joseph, R (1990)ATP: citrate lyase of Rhodotorula gracilis: purification and properties Biochim Biophys Acta 1033, 23–30.

8 Rangasamy, D & Ratledge, C (2000)Compartmentation of ATP: citrate lyase in plants Plant Physiol 122, 1225–1230.

9 Shiba, H., Kawasumi, T., Igarashi, Y., Kodama, T & Minoda, Y (1985)The CO 2 assimilation via the reductive tricarboxylic acid cycle in an obligately autotrophic, aerobic hydrogen-oxidizing bacterium Hydrogenobacter thermophilus Arch Microbiol 141, 198–203.

10 Schauder, R., Widdel, F & Fuchs, G (1987)Carbon assimilation pathways in sulfate-reducing bacteria II Enzymes of a reductive citric acid cycle in the autotrophic Desulfobacter hydrogenophilus Arch Microbiol 148, 218–225.

11 Beh, M., Strauss, G., Huber, R., Stetter, K.-O & Fuchs, G (1993) Enzymes of the reductive citric acid cycle in the autotrophic eubacterium Aquifex pyrophilus and in the archaebacterium Thermoproteus neutrophilus Arch Microbiol 160, 306–311.

12 Antranikian, G., Herzberg, C & Gottschalk, G (1982)Char-acterization of ATP citrate lyase from Chlorobium limicola.

J Bacteriol 152, 1284–1287.

13 Ishii, M., Igarashi, Y & Kodama, T (1989)Purification and characterization of ATP: citrate lyase from Hydrogenobacter thermophilus TK-6 J Bacteriol 171, 1788–1792.

14 Wahlund, T.M & Tabita, F.R (1997)The reductive tricarboxylic acid cycle of carbon dioxide assimilation: initial studies and pur-ification of ATP-citrate lyase from the green sulfur bacterium Chlorobium tepidum J Bacteriol 179, 4859–4867.

15 Kanao, T., Fukui, T., Atomi, H & Imanaka, T (2001)ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola is

a heteromeric enzyme composed of two distinct gene products Eur J Biochem 268, 1670–1678.

16 Kanao, T., Kawamura, M., Fukui, T., Atomi, H & Imanaka, T (2002)Characterization of isocitrate dehydrogenase from the green sulfur bacterium Chlorobium limicola A carbon dioxide-fixing enzyme in the reductive tricarboxylic acid cycle Eur J Biochem 269, 1926–1931.

17 Takeda, Y., Suzuki, F & Inoue, H (1969)ATP citrate lyase (citrate-cleavage enzyme) Methods Enzymol 13, 153–160.

18 Rashid, N., Morikawa, M., Nagahisa, K., Kanaya, S & Imanaka,

T (1997)Characterization of a RecA/RAD51 homologue from the hyperthermophilic archaeon Pyrococcus sp KOD1 Nucleic Acids Res 25, 719–726.

19 Segel, I.H (1993) Enzyme Kinetics: Behavior and Analysis of Rapid

Equilibrium and Steady-State Enzyme Systems Wiley Classics

Library Edition, New York.

20 Inoue, H., Suzuki, F., Tanioka, H & Takeda, Y (1968)Studies on

ATP citrate lyase of rat liver III The reaction mechanism.

J Biochem 63, 89–100.

21 Pearson, P.H & Bridger, W.A (1975)Catalysis of a step of the

overall reaction by the a subunit of Escherichia coli succinyl

coenzyme A synthetase J Biol Chem 250, 8524–8529.

22 Majumdar, R., Guest, J.R & Bridger, W.A (1991)Functional

consequences of substitution of the active site (phospho)histidine

residue of Escherichia coli succinyl-CoA synthetase Biochim.

Biophys Acta 1076, 86–90.

23 Wells, T.N.C (1991)ATP-citrate lyase from rat liver

Character-isation of the cytryl-enzyme complexes Eur J Biochem 199,

163–168.

24 Tuha´cˇkova´, Z & Krˇiva´nek, J (1996)GTP, a nonsubstrate of ATP

citrate lyase, is a phosphodonor for the enzyme histidine

auto-phosphorylation Biochem Biophys Res Commun 218, 61–66.

25 Joyce, M.A., Fraser, M.E., James, M.N.G., Bridger, W.A &

Wolodko, W.T (2000)ADP-binding site of Escherichia coli

suc-cinyl-CoA synthetase revealed by x-ray crystallography

Bio-chemistry 39, 17–25.

26 Johnson, J.D., Muhonen, W.W & Lambeth, D.O (1998)Char-acterization of the ATP- and GTP-specific succinyl-CoA synthe-tases in pigeon The enzymes incorporate the same a-subunit.

J Biol Chem 273, 27573–27579.

27 Pierce, M.W., Palmer, J.L., Keutmann, H.T & Avruch, J (1981) ATP-citrate lyase Structure of a tryptic peptide containing the phosphorylation site directed by glucagon and the cAMP-depen-dent protein kinase J Biol Chem 256, 8867–8870.

28 Ramakrishna, S., Pucci, D.L & Benjamin, W.B (1981)ATP-citrate lyase kinase and cyclic AMP-dependent protein kinase phosphorylate different sites on ATP-citrate lyase J Biol Chem.

256, 10213–10216.

29 Ramakrishna, S., D’Angelo, G & Benjamin, W.B (1990) Sequence of sites on ATP-citrate lyase and phosphatase inhibitor 2-phosphorylated by multifunctional protein kinase (a glycogen synthase kinase 3 like kinase) Biochemistry 29, 7617–7624.

30 Hughes, K., Ramakrishna, S., Benjamin, W.B & Woodgett, J.R (1992)Identification of multifunctional ATP-citrate lyase kinase

as the a-isoform of glycogen synthase kinase-3 Biochem J 288, 309–314.

31 Inoue, H., Suzuki, F., Fukunishi, K., Adachi, K & Takeda, Y (1966)Studies on ATP citrate lyase of rat liver I Purification and some properties J Biochem 60, 543–553.