Construction of new forms of pyruvate carboxylase to assess the allosteric regulation by acetyl-CoA

M.Nurul Islam, Shinji Sueda1 and Hiroki Kondo

Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Kawazu 680-4, Iizuka 820-8502, Japan

1 To whom correspondence should be addressed. E-mail: sueda{at}bio.kyutech.ac.jp


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The single polypeptide chain of Bacillus thermodenitrificans pyruvate carboxylase (PC) is composed of the biotin carboxylase (BC), carboxyl transferase (CT) and biotin carboxyl carrier protein (BCCP) domains from the amino terminus. This polypeptide chain was divided into two between the CT and BCCP domains. The resulting proteins, PC-(BC + CT) and PC-(BCCP), were expressed in Escherichia coli separately, purified to homogeneity and characterized. PC-(BC + CT) was 4% as active as native PC in the carboxylation of pyruvate with PC-(BCCP) as substrate with a Km of 39 µM. Moreover, acetyl-CoA stimulated the carboxylation of PC-(BCCP) about 3-fold, whereas it was without effect in the corresponding reaction with free biotin. In addition to these engineered proteins, another form of enzyme was also constructed in which the BC domain of B.thermodenitrificans PC was replaced with the BC subunit of Aquifex aeolicus PC, whose activity is independent of acetyl-CoA. The resulting chimera was about 7% as active as native PC, but its activity was independent of acetyl-CoA. On the basis of these observations, the mechanism by which acetyl-CoA regulates the reaction of PC is discussed.

Keywords: acetyl-CoA/biotin/chimera/pyruvate carboxylase


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pyruvate carboxylase (PC) (EC 6.4.1.1 [EC] ) is a biotin-dependent enzyme and is involved in gluconeogenesis by mediating carboxylation of pyruvate into oxalacetate (Utter and Keech, 1960Go). PC is distributed in many eukaryotes and also in some prokaryotes in two different forms (Barden et al., 1975Go; Jitrapakdee and Wallace, 1999Go). One form, found in eukaryotes and some prokaryotes, is made up of a single polypeptide chain of about 1200 amino acids. The other form, found only in prokaryotes, consists of two polypeptide chains with total amino acid residues similar to those of the former (Barden et al., 1975Go). The PC reaction is believed to proceed in two steps, just like those of other biotin-dependent carboxylases such as acetyl-CoA carboxylase (ACC): enzyme-bound biotin is carboxylated first by bicarbonate and ATP and the carboxyl group bound temporarily on biotin is subsequently transferred to pyruvate (Wood and Barden, 1977Go; Attwood, 1995Go):

(1)

(2)

Thus, PC carries at least three functional domains: biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), which mediates the first partial reaction (Equation 1) and carboxyl transferase (CT), which catalyzes the second partial reaction (Equation 2). The BC domain is located at the amino terminus of the single polypeptide chain of PC, followed by the CT domain with the biotin-carrying domain residing in the carboxyl terminus (Lim et al., 1988Go). In the subunit-type PC, the polypeptide chain is divided between the BC and CT domains (Mukhopadhyay et al., 1998Go). Acetyl-CoA and aspartate modulate the activity of the former class of PC allosterically, but the latter class of PC is insensitive to acetyl-CoA (Cazzulo and Stoppani, 1968Go; Ashman et al., 1972Go; Libor et al., 1978Go; Mukhopadhyay et al., 1998Go; Jitrapakdee and Wallace, 1999Go). Because of the lack of three-dimensional structural information, the detailed mechanism of carboxylation by PC and its allosteric regulation remain largely obscure. Hence, protein engineering approaches would be useful to unravel the mechanism of reaction and regulation of this enzyme. Here, PC from Bacillus thermodenitrificans was engineered in such a way as to divide the single polypeptide chain into two at the boundary of the CT and BCCP domains (Figure 1). The resulting two proteins, PC-(BC + CT) and PC-(BCCP), were purified and characterized. Together with it, a chimeric PC was also constructed by replacing the BC domain of B.thermodenitrificans PC with the BC subunit of Aquifex aeolicus PC, whose activity is independent of acetyl-CoA. On the basis of the kinetic properties of these engineered proteins, the mechanism of acetyl-CoA activation of PC is discussed.



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Fig. 1. Schematic presentation of B.thermodenitrificans PC and engineered proteins, PC-(BC + CT), PC-(BCCP), PC-(aaBC) and chimeric PC. It is noted that the PC-(aaBC) and the BC domain of chimeric PC derive from A.aeolicus. The N- and C-terminal boundaries of each domain are shown by the residue numbers. The initiating methionine was introduced to PC-(BCCP) as an additional residue. The arrow denotes the nicked position in PC-(BCCP). The initiating methionine of PC-(CT + BCCP) (Sueda et al., 2004Go) was not introduced to chimeric PC.

 

    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

Inorganic salts and common organic chemicals were obtained from commercial sources. Avidin was purchased from ProZyme (San Leandro, CA) and acetyl-coenzyme A from Wako Pure Chemicals (Osaka, Japan). Reagents for genetic engineering such as restriction enzymes were purchased from Takara (Kyoto, Japan) and oligonucleotides were custom synthesized by Hokkaido Science (Sapporo, Japan). The TOPO TA cloning kit was supplied by Invitrogen (Carlsbad, CA).

Construction of over-expression plasmids for PC-(BC + CT) and PC-(BCCP)

The B.thermodenitrificans PC gene cloned in pBluescript vector (pPC) was the source of engineering of this enzyme (Kondo et al., 1997Go). Based on the reasoning described in the Results section, the gene was divided at the MluI site present within 3123–3128 bp from the 5'-terminus of the open reading frame (ORF). The polypeptide chain was separated into two at this site by introducing a stop codon or an initiation codon for the expression of BC plus CT and BCCP, respectively. An expression plasmid for PC-(BC + CT) was prepared as follows: a C-terminal fragment of PC of about 325 bp was PCR-amplified with pPC as template using the following primers: F1, 5'-ACGCGTCTAATACTTCGAG-3' and B1, 5'-GAGCTCTTATTTGGACAACTCC-3' (restriction enzyme sites are underlined). The forward primer, F1, harbored the MluI site present on the PC gene and an artificial stop codon (shown in bold) for the original GTC codon and the reverse primer, B1, harbored a SacI site present on the vector and a stop codon. PCR was run as follows: after heating at 94°C for 5 min, the following cycle was repeated 30 times: 94°C, 1 min; 55°C, 1 min; 72°C, 1.5 min; and finally heated at 72°C for 6 min. The PCR product was TA cloned into pCR2.1-TOPO and sequenced. The plasmid thus prepared was digested with MluI/SacI restriction enzymes and the resulting fragment was recloned into the MluI/SacI sites of pPC to give a new recombinant plasmid. To enhance expression of the desired protein, the promoter region of this recombinant was replaced with the high-expression promoter trc of pTrc99A vector in the same way as that reported previously (Sueda et al., 2004Go). The resulting plasmid allowed Escherichia coli to express the BC plus CT domain of PC at a high level.

The PC-(BCCP) expression plasmid was prepared as follows: a C-terminal fragment of PC of about 325 bp was PCR-amplified with pPC as the template using the following primers: BCCP1, 5'-CCATGGTCTACTTCGAGCTGAACG-3' and BCCP2, 5'-AAGCTTTTATTTGGACAACTCC-3' (restriction enzyme sites are underlined). The forward primer, BCCP1, harbored an NcoI site containing an intiation codon (shown in bold) and the reverse primer, BCCP2, harbored a HindIII site and a stop codon (shown in bold). The resulting PCR product was TA cloned and sequenced. The insert was excised from the plasmid and recloned into the NcoI/HindIII sites of pET-21d to produce a recombinant plasmid for PC-(BCCP). The number of amino acid residues and the calculated molecular mass of the proteins prepared are as follows: PC-(BC + CT), 1043 residues, 116 826 Da; PC-(BCCP), 105 residues, 11 523 Da.

Construction of an over-expression plasmid for chimeric PC

The ß subunit (aaBC: MW 50 kDa) of A.aeolicus PC was amplified by PCR in a way identical with that described previously (Kondo et al., 2004Go). The PCR product was TA cloned and sequenced. The insert was cut out of the vector and cloned into the pTrc99A vector to yield a recombinant plasmid, pPC-(aaBC), which served as the source of the BC domain of chimeric PC. Thus, an ~950 bp C-terminal fragment of A.aeolicus BC gene was PCR-amplified with pPC-(aaBC) as template and oligonucleotides BC1 and BC2 as primers: BC1, 5'-CCGCGGGCGGTGGTGGTA-3', primes on the coding strand and contains a SacII site (underlined); BC2, 5'-GGTACCGTGGTAAGCGGCTATACGAGCCGA-3' primes on the non-coding strand and carries a KpnI site (underlined) in place of the stop codon. The resulting PCR product was TA cloned and sequenced. The insert was excised from the plasmid and recloned into the SacII/KpnI sites of pPC-(aaBC), to give pPC-(aaBC1). Likewise, an ~400 bp N-terminal fragment of the CT domain of B.thermodenitrificans PC was amplified with pPC-(CT + BCCP) (Sueda et al., 2004Go) as template using the following primers: CT1, 5'-GGTACCGCACGCCGGAAAGACCGCG-3' and CT2, 5'-CCGATCCCACGGATCCTCTTTTAAAAAGCG-3' (restriction enzyme sites are underlined). The forward primer, CT1, harbored a KpnI site introduced for gene manipulation and the reverse primer, CT2, harbored a BamHI site present on the PC-(CT + BCCP) gene. It is noted that the KpnI site was placed at the codons for the last two residues of BC so as not to change the amino acids. The resulting fragment was TA cloned and sequenced, then recloned into the KpnI/BamHI sites of pPC-(aaBC1) to yield pPC-(aaBC1 + CT1). The C-terminal fragment of PC, excised from the plasmid pPC-(CT + BCCP) with BamHI/PstI, was recloned into the same sites of pPC-(aaBC1 + CT1) to yield a recombinant plasmid, pchPC for chimeric PC. The number of amino acid residues and the calculated molecular mass of the chimeric protein prepared were 1158 residues and 129 685 Da, respectively.

Construction of an over-expression plasmid for birA

The coding region of the birA gene (966 bp) of E.coli was amplified by PCR in one step with the following primers: birA1, 5'-CATATGAAGGATAACACCGTGCCACTG-3'; birA2, 5'-CTCGAGTTATTTTTCTGCACTACGCAGGG-3' (restriction enzyme sites are underlined). PCR conditions were the same as those described above. The PCR product was purified by agarose gel electrophoresis before ligation into pCR2.1-TOPO. After confirming the correct DNA sequence, the coding region was excised from the plasmid and cloned into the NdeI/XhoI sites of pET-24a to produce pBirA.

Protein expression and purification

Escherichia coli JM109 transformed with one of the over-expression plasmids prepared as above except for pPC-(BCCP) and pBirA was grown in Luria-Bertani (LB) medium supplemented with 50 µg/ml ampicillin and 1 µg/ml D-biotin, where a biotin-binding domain was present. The two proteins, PC-(BCCP) and BirA, were expressed in E.coli BL21(DE3) (Novagen, Madison, WI) separately or simultaneously, following transformation with one or two of the plasmids prepared above. Transformants were grown in 1 l of LB medium in the presence of ampicillin (50 µg/ml) or kanamycin (30 µg/ml) or both. A fresh overnight culture (10 ml) from a single colony was used to inoculate 1 l of medium. The cultures were grown at 37°C for 8–10 h, then isopropyl ß-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and the cultures were incubated for an additional 8–10 h. The cells were harvested by centrifugation at 5000 r.p.m. (4200 g) for 10 min at 4°C. Proteins were purified according to the procedures previously described (Sueda et al., 2004Go; Yong-Biao et al., 2004Go). In brief, the harvested cells were disrupted by sonication and centrifuged. The proteins were purified by ammonium sulfate fractionation and N,N-diethylaminoethyl (DEAE)-cellulose chromatography (2 x 10 cm, Whatman, Maidstone, UK) and proteins were eluted with a linear gradient of 0–500 mM NaCl in 20 mM potassium phosphate (KPi), pH 7.0. The pooled fractions were concentrated and then subjected to gel filtration chromatography on Superdex 200 (Amersham, Piscataway, NJ). Chimeric PC and PC-(BCCP) were purified finally by monomeric avidin Sepharose affinity chromatography (Jitrapakdee et al., 1999Go). Other proteins were purified by anion-exchange chromatography on Mono Q HR 5/5 (Amersham). The protein concentration was determined spectroscopically from the amino acid composition.

In vitro and in vivo biotinylation of BCCP

In vitro biotinylation was carried out according to the procedures reported previously (Chapman-Smith et al., 1994Go, 1999Go). Unless stated otherwise, the reaction mixture contained 50 mM Tris–HCl (pH 8.0), 3 mM ATP, 5 mM MgCl2, 100 mM KCl, 0.5 mM biotin, a sufficient amount of apo-BCCP and biotin protein ligase, also called BirA. The reaction was initiated by the addition of biotin protein ligase and incubated at 37°C overnight, then the reaction mixture was applied to a monomeric avidin Sepharose affinity column and finally eluted with buffer containing 1 mg/ml biotin. In vivo biotinylation was performed by the co-expression of acceptor protein and biotin protein ligase in the presence of free biotin (1 µg/ml) (Chapman-Smith et al., 1994Go).

Pyruvate carboxylase assays

Pyruvate carboxylase was assayed in the direction of oxalacetate formation by coupling the reaction with malate dehydrogenase according to the methods described previously (Modak and Kelly, 1995Go). All assays were carried out at 30°C and the reaction mixture contained the following components, unless stated otherwise: 100 mM Tris–HCl (pH 8.0), 2 mM ATP, 5 mM MgCl2, 100 mM KCl, 10 mM pyruvate, 100 mM NaHCO3, 0.1 mM acetyl-CoA, 0.15 mM NADH, 5 units of malate dehydrogenase and 20–30 µg of PC-(BC + CT) or chimeric PC in 1 ml. For PC-(BC + CT) assay, 100 mM biotin or 100 µM BCCP was added to the above reaction mixture. The Michaelis constants (Km) of PC-(BC + CT) for ATP, bicarbonate, pyruvate, biotin or BCCP were calculated according to the procedure reported previously (Sueda et al., 2004Go). The Km values of chimeric PC for ATP, bicarbonate and pyruvate were determined analogously except that 5 mM rather than 2 mM fixed ATP was employed, as substrate inhibition for ATP was not apparent in this system. To evaluate allosteric cooperativity, PC activity was determined by varying the acetyl-CoA concentration from 0 to 200 µM with fixed concentrations of 10 mM pyruvate, 100 mM bicarbonate and 2 mM ATP; 100 µM PC-(BCCP) was used in the reaction mixture of PC-(BC + CT).

ATP cleavage assays in the absence of pyruvate

These assays were carried out at 30°C as described previously (Attwood and Graneri, 1992Go) using pyruvate kinase and lactate dehydrogenase as coupling enzymes to monitor MgADP formation. The reaction mixture contained the following components, unless stated otherwise: 100 mM Tris–HCl (pH 8.0), 2 mM ATP, 5 mM MgCl2, 100 mM KCl, 100 mM NaHCO3, 0.1 mM acetyl-CoA, 0.5 mM phosphoenol pyruvate, 0.15 mM NADH, 5 units of lactate dehydrogenase, 5 units of pyruvate kinase and 10–40 µg of PC-(BC + CT) or chimeric PC or 200 µg of PC-(aaBC) in 1 ml. For PC-(BC + CT) assay, 100 mM biotin or 100 µM BCCP was added to the above reaction mixture and for PC-(aaBC) assay, 100 mM biotin was added. The kinetic parameters, Km and Vmax values, of PC-(BC + CT) for biotin were determined by varying the biotin concentration from 0 to 100 mM with fixed concentrations of ATP (2 mM) and bicarbonate (100 mM) and the Km and Vmax for BCCP were determined by varying the BCCP concentration from 0 to 175 µM at fixed concentrations of ATP (2 mM) and bicarbonate (100 mM). Again, the kinetic parameters, Km and Vmax, for the ATPase reaction of PC-(aaBC) were determined according to the procedure described previously (Sueda et al., 2004Go).

To assess the binding of acetyl-CoA to PC-(BC), PC-(aaBC) and chimeric PC, their ATP cleavage activity was determined by varying the acetyl-CoA concentration from 0 to 200 µM with fixed concentrations of ATP (2 mM) and bicarbonate (100 mM). Free biotin (100 mM) was also present in the reaction mixture in the PC-(BC) and PC-(aaBC) systems.

Oxamate-induced oxalacetate decarboxylase assays

Oxalacetate decarboxylase activity of PC-(BC + CT) and chimeric PC was measured with oxamate as stimulant according to the procedures reported previously (Attwood and Cleland, 1986Go). The reactions were monitored by measuring the formation of pyruvate, which was then reduced to lactate by lactate dehydrogenase and the concomitant oxidation of NADH was monitored at 340 nm. All assays were performed at 30°C and the reaction mixture contained the following components, unless stated otherwise: 100 mM Tris–HCl (pH 8.0), 5 mM MgCl2, 100 mM KCl, 0.2 mM oxalacetate, 0.1 mM acetyl-CoA, 1 mM oxamate, 0.15 mM NADH, 5 units of lactate dehydrogenase and 30–60 µg of PC-(BC + CT) or chimeric PC in 1 ml. For PC-(BC + CT) assay, 100 mM biotin or 100 µM BCCP was added to the above reaction mixture. The reactions were started by the addition of PC-(BC + CT) or chimeric PC but, prior to the addition, a background rate of oxalacetate decarboxylation was established and this was subtracted from the rate in the presence of enzyme.

The kinetic parameters of PC-(BC + CT) for biotin were determined by varying its concentration from 0 to 100 mM at fixed concentrations of oxamate (1 mM) and oxalacetate (0.1 mM). Again, the kinetic parameters for BCCP were determined by varying its concentration from 0 to 200 µM at fixed concentrations of oxamate (1 mM) and oxalacetate (0.1 mM).


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Engineering of the pyruvate carboxylase gene

Although it is difficult to know exactly the boundary of the CT and BCCP domains of B.thermodenitrificans PC (1147 residues), it seemed certain that the CT domain ends before amino acid number around 940. For one thing, sequence homology is hardly seen in this region among PCs from various sources. Hence this PC gene was divided into two at the MluI site present around 3125 bp from the 5' end of ORF (Kondo et al., 1997Go). The upstream fragment (3129 bp) was supposed to harbor the BC and CT domains and the downstream fragment (312 bp) the BCCP domain (Figure 1). The genes for the two fragments were cloned separately into expression vectors and upon transformation of E.coli with either of the resulting recombinants, the two proteins were over-expressed successfully in soluble form and purified to near homogeneity by conventional methods (Figure 2). The yields were typically 8 and 30 mg for PC-(BC + CT) and PC-(BCCP), respectively, from a 2 l cell culture. The integrity of these proteins was assessed by N-terminal sequencing and it was found that PC-(BC + CT) was roughly a 1:1 mixture of proteins with and without the initiating methionine. The N-terminal sequence of PC-(BCCP) was found to be ADRTNPNHIA, i.e. the N-terminal 29 residues had been lost, leaving a 76-residue protein. This truncated PC-(BCCP) is referred to simply as BCCP hereafter and used throughout the present paper.



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Fig. 2. SDS–PAGE of engineered proteins. (A) SDS–PAGE with 10% polyacrylamide and 0.5 µg each of the proteins: M, high molecular weight markers; lane 1, PC-(BC + CT); lane 2, chimeric PC. (B) SDS–PAGE with 15% polyacrylamide and 0.5 µg of protein: M, low molecular weight markers; lane 1, PC-(BCCP).

 
Chimeric PC was prepared by replacing the BC domain of B.thermodenitrificans PC with the BC subunit of A.aeolicus PC. Thus, the 472-residue BC subunit of A.aeolicus PC was joined to the 686-residue PC-(CT + BCCP) (Sueda et al., 2004Go) to produce the 1158-residue chimeric PC, which is comparable in length with that of parent PC, 1147. It is noted that the C-terminal 21 residues of the BC subunit of A.aeolicus PC were disordered in its crystal structure, making only the rest of the protein (451 residues) visible (Kondo et al., 2004Go). Escherichia coli harboring the recombinant plasmid expressed the desired protein at a high level in soluble form, which was purified to homogeneity by conventional methods including avidin affinity chromatography with a yield of 10 mg from a 2 l culture (Figure 2). The N-terminal sequence was consistent with that deduced from the DNA sequence (Kondo et al., 2004Go).

Biotinylation of BCCP

It was found that BCCP prepared above was scarcely biotinylated; only 1 mg of the biotinylated form was obtained from 30–40 mg of BCCP purified from a 2 l culture. This observation was rather unexpected, as over-expressed PC from the same source and chimeric PC were biotinylated considerably under identical conditions (Sueda et al., 2004Go; Yong-Biao et al., 2004Go). To circumvent this, in vitro and in vivo biotinylation of apo BCCP was attempted. Thus, apo BCCP was first biotinylated with isolated E.coli holocarboxylase synthetase. It was found that in vitro biotinylation proceeded only very slowly and it was not practical to obtain milligram quantities of holo-BCCP by this method. To biotinylate BCCP in vivo, holocarboxylase synthetase and PC-(BCCP) were co-expressed in E.coli (Chapman-Smith et al., 1994Go) and, although the biotinylation rate was still small, holo-BCCP could be obtained following avidin affinity chromatography in quantities sufficient for subsequent kinetic studies.

Pyruvate carboxylase activity of PC-(BC + CT) and chimeric PC

It was found that the divided protein, PC-(BC + CT), is as capable as native PC of mediating pyruvate carboxylation in the presence of free D-biotin or holo-BCCP, suggesting that its three-dimensional structure remains largely intact even in the absence of the BCCP domain. The enzymic activity of PC-(BC + CT) determined at 30°C in the presence of various concentrations of pyruvate, ATP, bicarbonate and biotin or BCCP is shown in Figure 3. The Michaelis constants, Km, for the three substrates in the carboxylation of pyruvate by PC-(BC + CT) with free biotin and by chimeric PC were determined. The Km values for pyruvate of PC-(BC + CT) and chimeric PC were 0.28 ± 0.03 and 0.25 ± 0.02 mM, respectively, which were virtually identical with those of native PC (Sueda et al., 2004Go), but the Km for bicarbonate of chimeric PC (3.09 ± 0.46 mM) decreased 10-fold from that of native PC, presumably because the BC subunit of A.aeolicus PC was used (see below). The Km value of PC-(BC + CT) for bicarbonate was 22.1 ± 1.9 mM, which was nearly identical with that of native PC. In the kinetic analysis for ATP of PC-(BC + CT), substrate inhibition was manifest at high ATP concentrations just like in native PC and two kinds of data analysis were therefore exploited. The Km values of PC-(BC + CT) thus obtained for ATP with or without substrate inhibition taken into account were 1.01 ± 0.09 and 0.64 ± 0.06 mM, respectively, which were nearly identical with those of native PC in either of the data analyses (Sueda et al., 2004Go). Interestingly, substrate inhibition at high ATP concentrations was not evident with chimeric PC and the Km value (0.25 ± 0.01 mM) decreased to half.



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Fig. 3. Kinetic analysis for the pyruvate carboxylation reaction of PC-(BC + CT) in the presence of free biotin or BCCP. PC-(BC + CT) activity was assayed in 100 mM Tris–HCl (pH 8.0) containing 5 mM MgCl2, 100 mM KCl, 0.1 mM acetyl-CoA, 0.15 mM NADH, 5 units of malate dehydrogenase and various concentrations of pyruvate, ATP, bicarbonate, biotin or BCCP at 30°C. (A) Pyruvate was the variable substrate (0 to 10 mM) with 2 mM ATP, 100 mM bicarbonate and 100 mM biotin; the Km for pyruvate was 0.28 ± 0.03 mM and Vmax 24.4 ± 0.74 min–1. (B) ATP was the variable substrate (0–5 mM) with 10 mM pyruvate, 100 mM bicarbonate and 100 mM biotin. In the kinetics for ATP, substrate inhibition was evident and therefore two different kinds of analysis were made for the data obtained. Kinetic parameters determined with the data from 0 to 1.0 mM on the basis of the simple Michaelis–Menten equation were Km 0.64 ± 0.06 mM and Vmax 36.0 ± 1.8 min–1, whereas those determined from the data from 0 to 5.0 mM on the basis of considering substrate inhibition were Km 1.01 ± 0.09 mM and Vmax 51.9 ± 3.2 min–1. (C) Bicarbonate was the variable substrate (0.5–100 mM) with 10 mM pyruvate, 2 mM ATP and 100 mM biotin; the Km for bicarbonate was 22.1 ± 1.9 mM and Vmax 29.0 ± 0.9 min–1. (D) Biotin was the variable substrate (0–100 mM) with 10 mM pyruvate, 100 mM bicarbonate and 2 mM ATP; the Km for biotin was 23.2 ± 1.4 mM and Vmax 28.3 ± 0.6 min–1. (E) BCCP was the variable substrate (0–175 µM) with 10 mM pyruvate, 100 mM bicarbonate and 2 mM ATP; the Km for BCCP was 0.039 ± 0.003 mM and Vmax 60.6 ± 1.7 min–1. In each case, the kinetic parameters and their standard errors were determined by non-linear regression analysis.

 
The pyruvate carboxylase activity of native PC and PC-(BC + CT) was modulated by acetyl-CoA sigmoidally, as shown in Figure 4A, providing evidence for the allosteric nature of this modulation. These data were analyzed by the Hill equation to yield apparent n values of 3.0 and 3.2 for native PC and PC-(BC + CT), respectively (Figure 4B). In other words, cooperative binding of acetyl-CoA does not change significantly between the two enzymes. By contrast, the dissociation constant, KD, for acetyl-CoA of native PC, 44.0 µM, was considerably smaller than that of PC-(BC + CT), 56.2 µM. The PC activity of PC-(BC + CT) with BCCP as substrate was enhanced about 3-fold upon addition of acetyl-CoA, whereas it was without effect in the same reaction with free biotin as substrate (Table I). Again, the PC activity of chimeric PC was determined by varying the acetyl-CoA concentration, but it was independent of the ligand over the concentration range studied (0–200 µM) (Table I). It is, nevertheless, noteworthy that the activity of chimeric PC in the absence of acetyl-CoA was about 20 times higher than that of native PC.



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Fig. 4. (A) Effect of acetyl-CoA concentration on PC activity of native PC and PC-(BC + CT). PC activity was determined by varying the acetyl-CoA concentration (0–200 µM) with fixed concentrations of pyruvate (10 mM), bicarbonate (100 mM) and ATP (2 mM). BCCP (100 µM) was also present in the PC-(BC + CT) system. Data with closed circles and open circles represent native PC and PC-(BC + CT), respectively. (B) Hill plots for the data shown in (A). Symbols v and Vmax indicate the reaction rate and the maximum rate, respectively.

 

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Table I. Effect of 0.1 mM acetyl-CoA on pyruvate carboxylation reaction at pH 8.0 and 30°C

 
ATP cleavage activity of engineered proteins

PC is known to catalyze the ATP cleavage reaction in the absence of pyruvate (Attwood and Graneri, 1992Go) and hence it is possible to study the reaction of BC (Equation 1) independently of the CT reaction (Equation 2) by measuring this activity. The ATPase activity of PC-(BC + CT), determined under essentially the same conditions as the complete reaction except for the omission of pyruvate, also increased about 2.5-fold with BCCP in the presence of acetyl-CoA from that in its absence, but the activity was virtually the same with free biotin, regardless of the presence or absence of acetyl-CoA (Table II). The ATP cleavage reaction of chimeric PC did not change with acetyl-CoA concentration over the range adopted (0–200 µM), just like that of its overall reaction (Tables I and II). Again, the ATP cleavage activity of PC-(BC) and PC-(aaBC) with free biotin was not affected by acetyl-CoA over the concentration range adopted (0–200 µM).


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Table II. Effect of 0.1 mM acetyl-CoA on ATP cleavage reaction at pH 8.0 and 30°C

 
According to the proposed reaction mechanism for BC (Knowles, 1989Go; Attwood, 1995Go), the ATPase activity of PC-(aaBC) was dependent on three substrates and the activity in the absence of biotin is ~5% of the maximum in its presence. The Km value for bicarbonate was 2.88 ± 0.36 mM, which was nearly identical with that for the complete reaction of chimeric PC, and the Km value for biotin was 17.2 ± 1.1 mM. For ATP, substrate inhibition was observed just like in PC-(BC + CT) and the Km values evaluated by analysis of the data without and with substrate inhibition were 0.044 ± 0.001 and 0.058 ± 0.004 mM, respectively, which were considerably smaller than that of chimeric PC. As expected from the property of subunit type PCs (Cazzulo and Stoppani, 1968Go; Ashman et al., 1972Go; Libor et al., 1978Go), the ATPase activity of PC-(aaBC) was virtually unchanged in the presence and absence of acetyl-CoA (Table II).

In our previous study (Sueda et al., 2004Go), an engineered protein PC-(BC) was prepared by dividing the single polypeptide chain of B.thermodenitrificans PC at the boundary of the BC and CT domains and the ATPase activity of the resulting protein, determined with free biotin as substrate, was independent of acetyl-CoA (Table II). When studied again here but with BCCP as substrate by varying acetyl-CoA concentration from 0 to 200 µM, acetyl-CoA dependence was observed. The Hill coefficient, apparent n value, calculated by the Hill equation for acetyl-CoA and PC-(BC) was 2.0, which is considerably smaller than that of native PC and PC-(BC + CT). The KD value for acetyl-CoA of PC-(BC) with BCCP as substrate was 56.6 µM, which is identical with that of PC-(BC + CT) but considerably larger than that of native PC. The ATP cleavage activity of PC-(BC) with BCCP as substrate in the presence of the saturated concentration of acetyl-CoA was 4.3 times higher than that in its absence (Table II).

Oxamate-induced oxalacetate decarboxylase activity of PC-(BC + CT) and chimeric PC

It was reported that oxamate stimulates the decarboxylation of oxalacetate by PC (Attwood and Cleland, 1986Go). Hence it is possible to study the CT reaction (Equation 2) of PC separately from the BC reaction (Equation 1) with this assay (Attwood and Cleland, 1986Go). The oxamate-induced decarboxylation of oxalacetate by PC-(BC + CT) with BCCP, investigated by measuring the oxalacetate decarboxylase activity in the presence of a saturating concentration of oxamate (1 mM) at 30°C, was about 30 times higher than that in its absence (Table III), as in native PC. Such a phenomenon was also observed with free biotin as the substrate. Likewise, the oxalacetate decarboxylase activity of chimeric PC increased about 6-fold on addition of oxamate. The oxalacetate decarboxylase activity of PC-(BC + CT) and chimeric PC increased with increase in oxamate concentration up to 1 mM, followed by substrate inhibition. The effect of oxalacetate concentration on the decarboxylation reaction of these enzymes was also determined, where substrate inhibition was manifest at high concentrations of oxalacetate. Such a phenomenon was observed also for native PC (Sueda et al., 2004Go) and PC from chicken liver and was accounted for by competitive substrate inhibition (Attwood and Cleland, 1986Go). It is noted that the decarboxylation activity of PC-(BC + CT) and chimeric PC was virtually the same in the presence and absence of acetyl-CoA (Table III); therefore, the catalytic reaction of the CT domain appears to be independent of acetyl-CoA.


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Table III. Oxalacetate decarboxylation activity (min–1) of PC-(BC + CT) and chimeric PC in the presence or absence of 0.1 mM acetyl-CoA at pH 8.0 and 30°C

 
Substrate activity of BCCP in the reactions of PC-(BC + CT)

The kinetic parameters for the reactions of pyruvate carboxylation, ATP cleavage and oxalacetate decarboxylation of PC-(BC + CT) with BCCP or biotin as substrates are summarized in Table IV. The maximum velocity (Vmax) of pyruvate carboxylation with BCCP as substrate was about two times greater than that with biotin as substrate. More significantly, the Km for BCCP is 590-fold lower than that for free biotin. As a result, BCCP is 1270-fold more efficient as substrate than biotin in terms of the catalytic efficiency (Vmax/K). Again, the maximum velocity of ATP cleavage reaction with BCCP was 4.5 times greater than that with biotin, whereas the Km for BCCP was 620 times smaller than that for biotin. Hence the catalytic efficiency of ATP cleavage with BCCP as substrate was 2810 times greater than that with biotin. Similarly, the effect of BCCP on the oxalacetate decarboxylation reaction was studied, where the maximum velocity with BCCP was 2.4 times greater than that with biotin and the Km for BCCP was 200 times lower than that for biotin. Hence oxalacetate decarboxylation is 480 times catalytically more efficient with BCCP than with biotin as substrate. It is worth noting that the results obtained here are reminiscent of the BC and CT activity of E.coli ACC with respect to BCCP and free biotin as substrates (Fall et al., 1971Go; Blanchard et al., 1999Go).


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Table IV. Kinetic parameters for pyruvate carboxylation, ATP cleavage and oxalacetate decarboxylation reactions of PC-(BC + CT) in the presence of BCCP or free biotin at pH 8.0 and 30°Ca

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pyruvate is an end-product of glycolysis and metabolized further to various compounds. For example, it is converted to oxalacetate by carboxylation, acetyl-CoA by decarboxylative oxidation and lactate by hydrogenation. Some of the products of these reactions are transformed further: oxalacetate into aspartate by transamination and oxalacetate and acetyl-CoA into citrate by condensation. It is not surprising, therefore, to see that PC is regulated by some of these metabolites to partition pyruvate into available pathways. Nevertheless, the mechanism of this allosteric regulation of PC by these agents has remained largely obscure and to explore the mode of action of acetyl-CoA a protein engineering approach was taken in the present study.

As described above, Bacillus PC was divided between the CT and BCCP domains and a chimeric PC was constructed by replacing the BC domain of Bacillus PC with the BC subunit of Aquifex PC. In the former, the selection of the boundary of the CT and BCCP domains was rather arbitrary, and in order to save the activity of CT the N-terminal portion of the BCCP domain may have been sacrificed. Moreover, the PC-(BCCP) protein thus designed (105 residues) was nicked during expression at nearly one-third of the sequence from the N-terminus, leaving a 76-residue truncated protein. Nonetheless, it possessed most of the characteristics expected for the ‘native’ BCCP, as discussed below.

In Bacillus PC, the BCCP domain is fused with other domains and it is unable to evaluate its intrinsic affinity for them. The Km of 39 µM of PC-(BC + CT) for the 76-residue BCCP obtained experimentally was comparable to that of BC of E.coli ACC for BCCPs (the 87-residue protein formed by truncation in the middle of native BCCP, Km 160 µM) (Fall et al., 1971Go; Blanchard et al., 1999Go), suggesting that these two enzymes possess affinity for BCCP nearly of the same magnitude. In addition, the substrate activity of truncated PC-(BCCP) in pyruvate carboxylation was much higher than that of free biotin in terms of both Vmax and Km, 2- and 600-fold, respectively (Table IV). It is therefore obvious that the affinity of biotin is enhanced markedly by the addition of the 76-residue polypeptide chain. In addition to this enhanced affinity, the BCCP made the carboxylation of pyruvate susceptible to allosteric regulation by acetyl-CoA. Although the magnitude of activation of three times is modest compared with that of 280 times with native PC (Table I), the BCCP seemingly interacts with PC-(BC + CT) in a way in which free biotin can never do.

In the present study, it was found that acetyl-CoA affects both pyruvate carboxylation and ATPase reactions of PC-(BC + CT) in the presence of BCCP, but it is without effect in the same reactions with free biotin. Likewise, acetyl-CoA affected the ATPase reaction of PC-(BC) only in the presence of BCCP. By contrast, the oxalacetate decarboxylation reaction of PC-(BC + CT) was not dependent on acetyl-CoA even in the presence of BCCP. From these observations, it is obvious that acetyl-CoA affects the BC reaction of PC, but not the CT reaction. Judging from the difference in the effect of acetyl-CoA on PC activation between BCCP and free biotin, it is inferred that the peptide chain of the former interacts somehow with the acetyl-CoA binding site of BC to promote the catalytic reaction of BC. However, the effect of acetyl-CoA on the activation of the engineered protein with BCCP is considerably smaller than that of native PC. Hence another role, as proposed in our previous paper (Sueda et al., 2004Go), that the induction of the conformational change between the BC and CT domains of native PC should also be considered.

In the chimeric PC, both the carboxylation of pyruvate and ATPase activity were independent of acetyl-CoA, presumably because the BC domain deriving from Aquifex PC lacks the acetyl-CoA binding site or alternatively interfacing of the first and second partial reactions (Equations 1 and 2) does not occur properly. The third possibility would be that the conformation of the chimera is moderately proper already from the beginning for the reaction to occur, as inferred from its high basal activity (Table I).

From the Hill coefficients and dissociation constants, it seems certain that acetyl-CoA binds to the BC domain of PC, although the affinity for acetyl-CoA is partially impaired with PC-(BC) and PC-(BC + CT) from that of native PC, because of the truncation of the CT and/or BCCP domains. To identify the exact acetyl-CoA binding motif, information on the three-dimensional structure of PC-(BC) and native PC is essential and such an undertaking is under way in this laboratory.


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 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received November 5, 2004; revised January 31, 2005; accepted February 14, 2005.

Edited by Anthony Wilkinson





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