Regulation of an Escherichia coli/Mammalian Chimeric Carbamoyl-phosphate Synthetase
1998; Elsevier BV; Volume: 273; Issue: 47 Linguagem: Inglês
10.1074/jbc.273.47.31195
ISSN1083-351X
AutoresNisha Sahay, Hedeel I. Guy, Xin Liu, David R. Evans,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoCarbamoyl-phosphate synthetase (CPSase) consists of a 120-kDa synthetase domain (CPS) that makes carbamoyl phosphate from ATP, bicarbonate, and ammonia usually produced by a separate glutaminase domain. CPS is composed of two subdomains, CPS.A and CPS.B. Although CPS.A and CPS.B have specialized functions in intact CPSase, the separately cloned subdomains can catalyze carbamoyl phosphate synthesis. This report describes the construction of a 58-kDa chimeric CPSase composed of Escherichia coli CPS.A catalytic subdomains and the mammalian regulatory subdomain. The catalytic parameters are similar to those of the E. coli enzyme, but the activity is regulated by the mammalian effectors and protein kinase A phosphorylation. The chimera has a single site that binds phosphoribosyl 5′-pyrophosphate (PRPP) with a dissociation constant of 25 μm. The dissociation constant for UTP of 0.23 mm was inferred from its effect on PRPP binding. Thus, the regulatory subdomain is an exchangeable ligand binding module that can control both CPS.A and CPS.B domains, and the pathway for allosteric signal transmission is identical in E. coli and mammalian CPSase. A deletion mutant that truncates the polypeptide within a postulated regulatory sequence is as active as the parent chimera but is insensitive to effectors. PRPP and UTP bind to the mutant, suggesting that the carboxyl half of the subdomain is essential for transmitting the allosteric signal but not for ligand binding. Carbamoyl-phosphate synthetase (CPSase) consists of a 120-kDa synthetase domain (CPS) that makes carbamoyl phosphate from ATP, bicarbonate, and ammonia usually produced by a separate glutaminase domain. CPS is composed of two subdomains, CPS.A and CPS.B. Although CPS.A and CPS.B have specialized functions in intact CPSase, the separately cloned subdomains can catalyze carbamoyl phosphate synthesis. This report describes the construction of a 58-kDa chimeric CPSase composed of Escherichia coli CPS.A catalytic subdomains and the mammalian regulatory subdomain. The catalytic parameters are similar to those of the E. coli enzyme, but the activity is regulated by the mammalian effectors and protein kinase A phosphorylation. The chimera has a single site that binds phosphoribosyl 5′-pyrophosphate (PRPP) with a dissociation constant of 25 μm. The dissociation constant for UTP of 0.23 mm was inferred from its effect on PRPP binding. Thus, the regulatory subdomain is an exchangeable ligand binding module that can control both CPS.A and CPS.B domains, and the pathway for allosteric signal transmission is identical in E. coli and mammalian CPSase. A deletion mutant that truncates the polypeptide within a postulated regulatory sequence is as active as the parent chimera but is insensitive to effectors. PRPP and UTP bind to the mutant, suggesting that the carboxyl half of the subdomain is essential for transmitting the allosteric signal but not for ligand binding. carbamoyl-phosphate synthetase or its activity the CPSase synthetase domain that catalyzes bicarbonate activation the CPSase synthetase domain that catalyzes the phosphorylation of carbamate A2, and A3, the subdomains of CPS.A B2, and B3, the subdomains of CPS.B the multifunctional protein having glutamine-dependent carbamoyl phosphate synthetase, aspartate transcarbamoylase, anddihydroorotase activities the synthetase domain or subunit of carbamoyl-phosphate synthetase the amidotransferase or glutaminase domain or subunit of carbamoyl-phosphate synthetase phos- phoribosyl 5′-pyrophosphate. Escherichia coli carbamoyl-phosphate synthetase (CPSase1; EC 6.3.5.5) consists of a 40-kDa glutaminase subunit and a 120-kDa synthetase subunit (1Trotta P.P. Burt M.E. Haschemeyer R.H. Meister A. Proc. Natl. Acad. Sci. U. S. 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UMP is a feedback inhibitor, while ornithine, IMP, and NH3activate the enzyme. The elegant three-dimensional structure of E. coli carbamoyl-phosphate synthetase has recently been solved (15Thoden J.B. Holden H.M. Wesenberg G. Raushel F.M. Rayment I. Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (307) Google Scholar) to 2.8-Å resolution. Mammalian glutamine-dependent carbamoyl-phosphate synthetase catalyzes the first committed step in the de novopyrimidine biosynthetic pathway. The enzyme is part of a large multifunctional protein (17Shoaf W. Jones M. Biochem. Biophys. Res. Commun. 1971; 45: 796-802Crossref PubMed Scopus (50) Google Scholar, 18Shoaf W.T. Jones M.E. Biochemistry. 1973; 12: 4039-4051Crossref PubMed Scopus (143) Google Scholar, 19Mori M. Ishida H. Tatibana M. Biochemistry. 1975; 14: 2622-2630Crossref PubMed Scopus (79) Google Scholar, 20Coleman P.F. Suttle D.P. Stark G.R. J. Biol. 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Zimmermann B.H. Scully J.L. Kim H. Evans D.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 174-178Crossref PubMed Scopus (57) Google Scholar, 28Simmer J.P. Kelly R.E. Rinker Jr., A.G. Scully J.L. Evans D.R. J. Biol. Chem. 1990; 265: 10395-10402Abstract Full Text PDF PubMed Google Scholar, 29Bein K. Simmer J.P. Evans D.R. J. Biol. Chem. 1991; 266: 3791-3799Abstract Full Text PDF PubMed Google Scholar, 30Kim H. Kelly R.E. Evans D.R. J. Biol. Chem. 1992; 267: 7177-7184Abstract Full Text PDF PubMed Google Scholar) is organized into multiple, autonomously folded domains (Fig. 1), each with a distinct function. The CPSase activity of CAD, the major locus of regulation of the de novo pyrimidine biosynthetic pathway, is inhibited by UTP, activated by PRPP (31Hager S.E. Jones M.E. J. Biol. Chem. 1967; 242: 5667-5673Abstract Full Text PDF PubMed Google Scholar, 32Tatibana M. Ito K. J. Biol. Chem. 1969; 244: 5403-5413Abstract Full Text PDF PubMed Google Scholar, 33Levine R.L. Hoogenraad N.J. 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Biochemistry. 1992; 31: 1656-1664Crossref PubMed Scopus (35) Google Scholar, 39Stapleton M.A. Javid-Majd F. Harmon M.F. Hanks B.A. Grahmann J.L. Mullins L.S. Raushel F.M. Biochemistry. 1996; 35: 14352-14361Crossref PubMed Scopus (55) Google Scholar, 40Javid-Majd F. Stapleton M.A. Harmon M.F. Hanks B.A. Mullins L.S. Raushel F.M. Biochemistry. 1996; 35: 14362-14369Crossref PubMed Scopus (33) Google Scholar), we have made the surprising discovery (41Guy H.I. Evans D.R. J. Biol. Chem. 1996; 271: 13762-13769Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) that when cloned and expressed separately, each of the domains is functionally equivalent. CPS.A alone can catalyze both ATP-dependent partial reactions and ammonia-dependent carbamoyl-phosphate synthesis. The same results were obtained for the isolated CPS.B domain, except in this case, unlike the CPS.A domain, the activity is inhibited by UTP and activated by PRPP. The molecules are dimeric (Fig. 2), and while the monomers can each catalyze both partial reactions, the dimer (42Guy H.I. Schmidt B. Herve G. Evans D.R. J. Biol. Chem. 1998; 273: 14172-14178Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) is required to catalyze the overall biosynthetic reaction. The monomers in the homodimer probably have the same function as the two fused domains, CPS.A and CPS.B, in the intact molecule. Sequence homology (5Nyunoya H. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4629-4633Crossref PubMed Scopus (123) Google Scholar, 27Simmer J.P. Kelly R.E. Rinker Jr., A.G. Zimmermann B.H. Scully J.L. Kim H. Evans D.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 174-178Crossref PubMed Scopus (57) Google Scholar, 30Kim H. Kelly R.E. Evans D.R. J. Biol. Chem. 1992; 267: 7177-7184Abstract Full Text PDF PubMed Google Scholar, 43Evans D.R. Balon M.A. Biochim. Biophys. Acta. 1988; 953: 185-196Crossref PubMed Scopus (25) Google Scholar, 44Lim F. 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Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (307) Google Scholar), which revealed in addition that the A2 and B2 subdomains are each composed of two smaller subdomains. Allosteric effectors bind to the B3 subdomain at the extreme carboxyl end of the synthetase domain in E. coli CPSase (39Stapleton M.A. Javid-Majd F. Harmon M.F. Hanks B.A. Grahmann J.L. Mullins L.S. Raushel F.M. Biochemistry. 1996; 35: 14352-14361Crossref PubMed Scopus (55) Google Scholar, 47Rubio V. Cervera J. Lusty C.J. Bendala E. Britton H.G. Biochemistry. 1991; 30: 1068-1075Crossref PubMed Scopus (59) Google Scholar,59Bueso J. Lusty C.J. Rubio V. Biochem. Biophys. Res. Commun. 1994; 203: 1083-1089Crossref PubMed Scopus (17) Google Scholar, 60Cervera J. Bendala E. Britton H.G. Bueso J. Nassif Z. Lusty C.J. Rubio V. Biochemistry. 1996; 35: 7247-7255Crossref PubMed Scopus (30) Google Scholar, 61Cervera J. Conejero-Lara F. Ruiz-Sanz J. Galisteo M.L. Mateo P.L. Lusty C.J. Rubio V. J. Biol. 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Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (307) Google Scholar). Moreover, kinetic studies of the E. coli enzyme (66Braxton B.L. Mullins L.S. Raushel F.M. Reinhart G.D. Biochemistry. 1992; 31: 2309-2316Crossref PubMed Scopus (31) Google Scholar) showed that CPS.B is the major target of allosteric regulation. We have found (65Liu X. Guy H.I. Evans D.R. J. Biol. Chem. 1994; 269: 27747-27755Abstract Full Text PDF PubMed Google Scholar) that allosteric effectors also bind to the B3 region of CAD by constructing an interesting chimeric protein in which the E. coli CPSase regulatory domain (B3) was replaced by the B3 subdomain of CAD. The chimera was inhibited by UTP but not by the E. coli effector UMP and was activated by PRPP, a metabolite that has no effect on the E. coli enzyme. In this report, we describe the construction and the function of a chimeric CPSase consisting of E. coli CPSase A1 and A2 subdomains fused to the mammalian CPSase B3 subdomain. The 7.3-kilobase pair plasmid pHL2, containing the coding sequences for a chimeric molecule consisting of the complete E. coli CPSase domain with only the B3 regulatory domain replaced with the corresponding region of CAD, was constructed previously (65Liu X. Guy H.I. Evans D.R. J. Biol. Chem. 1994; 269: 27747-27755Abstract Full Text PDF PubMed Google Scholar) and is under the control of thecarB promoter. The E. coli strain RC50, defective in the carA and carB genes encoding the small and large subunits of E. coli CPSase, respectively, was kindly provided by Dr. Carol Lusty (Public Health Research Institute of the City of New York) (46Guillou F. Rubino S.D. Markovitz R.S. Kinney D.M. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8304-8308Crossref PubMed Scopus (37) Google Scholar). The recombinant proteins, which are expressed constitutively under the control of the carB promoter, were isolated from 200-ml cultures of transformed RC50 cells grown to stationary phase (46Guillou F. Rubino S.D. Markovitz R.S. Kinney D.M. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8304-8308Crossref PubMed Scopus (37) Google Scholar). The cells were resuspended in 2 ml of 50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm dithiothreitol, and 5% glycerol buffer and broken by sonication for 2 min at 4 °C. The sonicate was centrifuged at 10,000 × g for 20 min at 4 °C. Transformation was carried out by the Hanahan procedure (67Hanahan D. DNA Cloning: A Practical Approach.in: Glover D.M. IRL Press, New York1985Google Scholar). Restriction digests, ligations, and other DNA procedures were carried out using standard protocols (68Maniatis T. Fritsch E.R. Sambrook I. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). The plasmid pHE-A12eB3m encoding the E. coli CPS A1 and A2 and mammalian B3 subdomains (Fig. 1), was constructed 2In the original construct, the borders of the regulatory domain of CAD were defined (28Simmer J.P. Kelly R.E. Rinker Jr., A.G. Scully J.L. Evans D.R. J. Biol. Chem. 1990; 265: 10395-10402Abstract Full Text PDF PubMed Google Scholar, 30Kim H. Kelly R.E. Evans D.R. J. Biol. Chem. 1992; 267: 7177-7184Abstract Full Text PDF PubMed Google Scholar) by sequence homology and controlled proteolysis experiments. In the new construct reported here, an additional 25 residues were deleted from the amino end of the mammalian regulatory domain so that it corresponds more closely to the size of the regulatory domain described in the x-ray structure (15Thoden J.B. Holden H.M. Wesenberg G. Raushel F.M. Rayment I. Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (307) Google Scholar) of E. coli CPSase. Superscripts m and e refer to the origin of the domain, the mammalian and E.coli proteins, respectively. by reacting the plasmid, pHL2, with NspV (site in the E. colisequence, position 2,730) and StuI (site in the CAD sequence, position 3,892). The 4.3-kilobase pair fragment was then religated using T4 DNA ligase following treatment with the Klenow fragment of DNA polymerase. The 4.2-kilobase pair recombinant, pNS-A12eB3mΔ, was constructed in order to study the consequences of deleting the carboxyl end of the CAD regulatory domain in the chimera. The plasmid, pHE-A12eB3m was reacted with SalI (site in CAD, position 4,205) and EcoRI (site in the vector), and the ends were made flush using the Klenow fragment prior to religation. Protein concentrations were determined using the micro-BCA protein assay reagent kit from Pierce and by scanning Coomassie Blue-stained polyacrylamide gels. SDS-gel eletrophoresis was carried out on 10% polyacrylamide gels using the Laemmli procedure (69Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207516) Google Scholar). The gels were scanned using an HP Scan Jet 4C, UN-SCAN-IT gel Automated Digitizing System, and the concentration of the isolated proteins was determined by measuring the ratio of peak density to total density in a given lane and the total amount of protein applied to the gel. The background density was subtracted, and all measurements were made within the linear range of the densitometer. The recombinant protein was cross-linked using dimethyl suberimidate following the procedure (42Guy H.I. Schmidt B. Herve G. Evans D.R. J. Biol. Chem. 1998; 273: 14172-14178Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) used for intact CAD. The reaction mixture consisted of 0.03 mg of cross-linking reagent and 10–20 μg of protein in a total volume of 0.5 ml of 0.1 m N-ethylmorpholinacetic acid, pH 8.5. The reaction, which was carried out at room temperature, was initiated by the addition of the cross-linking reagent and quenched by the addition of 1 mglycine to a final concentration of 0.1 m. The extent of cross-linking was determined by SDS-polyacrylamide gel electrophoresis. Carbamoyl-phosphate synthetase activity was assayed using a radiometric procedure (20Coleman P.F. Suttle D.P. Stark G.R. J. Biol. Chem. 1977; 252: 6379-6385Abstract Full Text PDF PubMed Google Scholar, 22Mally M.I. Grayson D.R. Evans D.R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6647-6651Crossref PubMed Scopus (46) Google Scholar) as modified by Carrey et al. (35Shaw S.M. Carrey E.A. Eur. J. Biochem. 1992; 207: 957-965Crossref PubMed Scopus (26) Google Scholar) in which carbamoyl phosphate was trapped as acid-stable carbamoyl aspartate. The ATP concentration ranged between 0 and 5 mm, and 0.75 mm ATP, 10 mm MgCl2, 20 mm NH4Cl, and 12 mmNaHCO3 were used for all effector response curves. The protocol for phosphorylation was described previously by Carreyet al. (36Carrey E.A. Campbell D.G. Hardie D.G. EMBO J. 1985; 4: 3735-3742Crossref PubMed Scopus (58) Google Scholar) with slight modification (65Liu X. Guy H.I. Evans D.R. J. Biol. Chem. 1994; 269: 27747-27755Abstract Full Text PDF PubMed Google Scholar). The kinetic data were fit by least squares analysis (Micromath Scientist) to the Michaelis-Menten equation, the Hill equation, or the expressionv =V max[S]/(K m + S + S2/K i) which takes substrate inhibition into account. The procedure of Khorana (70Khorana H.G. Fernandes J.F. Kornberg A. J. Biol. Chem. 1958; 230: 941-948Abstract Full Text PDF PubMed Google Scholar) was used to synthesize [32P]phosphoribosyl-5′-pyrophosphate. Radiolabeled PRPP (18,000 cpm/nmol) is enzymatically synthesized from ribose-5′-phosphate and ATP (500 μCi of [γ-32P]ATP; Amersham Pharmacia Biotech) in a reaction catalyzed by PRPP synthetase, which was kindly provided by Robert Switzer (University of Illinois Urbana-Champaign). The specific radioactivity was determined by quantitating PRPP using an assay (18Shoaf W.T. Jones M.E. Biochemistry. 1973; 12: 4039-4051Crossref PubMed Scopus (143) Google Scholar) that measures the amount of [14C]CO2 produced from PRPP and [14C-COOH]orotidylate in the coupled reactions catalyzed by orotidylate PRPP transferase and OMP decarboxylase. The final concentrations in a 1-ml assay mixture were 0.1 m Tris-HCl, pH 7.5, 2 mm MgCl2, 100 μm[14C]orotate, and 1–20 nmol of PRPP. The reaction was initiated with 1–10 units of PRPP transferase-OMP decarboxylase mixed enzyme (Sigma). Following incubation for 1 h at room temperature, the reaction was quenched with 0.2 ml of 4 m perchloric acid. The [14C]carbon dioxide generated in the reaction was trapped in 0.2 ml of a 1:1 (v/v) ethylene glycol/ethanolamine solution and counted in a liquid scintillation counter. the binding of radiolabeled PRPP to protein was measured using the spin column procedure (72Penefsky H.S. Methods Enzymol. 1979; 56: 527-530Crossref PubMed Scopus (343) Google Scholar) that separates free-ligand and protein-bound ligand. The protein sample was incubated with [32P]PRPP for 15 min at 37 °C. The 150-μl reaction was then transferred to a 0.7 × 2.8-cm bed Nick spin column (Amersham Pharmacia Biotech). The protein-bound ligand was eluted by centrifugation at 1,000 × g for 4 min. Under these conditions, the volume of the eluant varied from 145 to 155 μl, and the recovery of the protein was 98–100%. The amount of bound PRPP was determined by liquid scintillation counting. The validity of the method was confirmed by measuring the binding of PRPP to CAD by continuous flow dialysis 3X. Liu, N. Sahay, G. Herve, and D. Evans, unpublished observations. and, for the chimeric protein, by equilibrium dialysis. For equilibrium dialysis, a 100-μl sample of the chimeric protein (0.61 mg/ml) was dialyzed against 0.5 ml of PRPP, at the indicated concentrations, in 50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm dithiothreitol, and 5% glycerol. After 24 h of equilibration, the PRPP concentration within the cell was determined by the PRPP assay described above. The same binding parameters were obtained by flow dialysis for CAD and by equilibrium dialysis for the chimeric protein as were obtained by the microcolumn method. Although the fraction of total ligand bound to the protein is low in the microcolumn method, the data are nevertheless reasonably accurate, since the column effectively separates free and bound ligand. The background counts in the eluted fraction obtained by applying the same amount of PRPP to the microcolumn in the absence of protein were quite low. For example, at 50 μm PRPP, the average value of the background for six experiments was 46 ± 6 cpm, compared with a typical value of 1,615 cpm in the presence of protein. At all PRPP concentrations, the signal:background ratio was at least 10. The dissociation constant for UTP was determined indirectly by measuring the PRPP binding in the presence of several different UTP concentrations. The dissociation constant was obtained using the expression ν½ = [PRPP]/([PRPP] +K PRPP(1 + [X]½/K UTP)), where [X]½ is the concentration of UTP required to displace half of the bound PRPP (ν½). K PRPP and [PRPP] are the measured PRPP dissociation constant and the concentration, respectively. We previously constructed (65Liu X. Guy H.I. Evans D.R. J. Biol. Chem. 1994; 269: 27747-27755Abstract Full Text PDF PubMed Google Scholar) a plasmid, pHL2, which codes for a chimeric protein in which the CPS B3 subdomain of theE. coli synthetase subunit was replaced by the corresponding subdomain of CAD (Fig. 1). A new plasmid, pHE-A12eB3m, that encodes the E. coli CPS A1 and A2 (residues 1–359) fused to the mammalian B3 subdomain (residues 1,298–1,461) was constructed by deleting theE. coli CPS A3, B1, and B2 subdomains from the chimeric plasmid, pHL2 (65Liu X. Guy H.I. Evans D.R. J. Biol. Chem. 1994; 269: 27747-27755Abstract Full Text PDF PubMed Google Scholar). When pHE-A12eB3m was transformed into the E. coli strain RC50, a uridine auxotroph that lacks the E. coli carA and carB genes, SDS-gel electr
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