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The Smallest Carbamoyl-phosphate Synthetase

1997; Elsevier BV; Volume: 272; Issue: 46 Linguagem: Inglês

10.1074/jbc.272.46.29255

ISSN

1083-351X

Autores

Hedeel I. Guy, Anne Bouvier, David R. Evans,

Tópico(s)

Enzyme Structure and Function

Resumo

Escherichia coli carbamoyl-phosphate synthetase (CPSase) is comprised of a 40-kDa glutaminase (GLN) and a 120-kDa synthetase (CPS) subunit. The CPS subunit consists of two homologous domains, CPS.A and CPS.B, which catalyze the two different ATP-dependent partial reactions involved in carbamoyl phosphate synthesis. Sequence similarities and controlled proteolysis experiments suggest that the CPS subdomains consist, in turn, of three subdomains, designated A1, A2, A3 and B1, B2, B3 for CPS.A and CPS.B, respectively. Previous studies of individually cloned CPS.A and CPS.B from E. coli and mammalian CPSase have shown that homologous dimers of either of these “half-molecules” could catalyze all three reactions involved in ammonia-dependent carbamoyl phosphate synthesis. Four smaller recombinant proteins were made for this study as follows: 1) A1-A2 in which the A3 subdomain was deleted from CPS.A, 2) B1-B2 lacking subdomain B3 of CPS.B, 3) the A2 subdomain of CPS.A, and 4) the B2 subdomain of CPS.B. When associated with the GLN subunit, A1-A2 and B1-B2 had both glutamine- and ammonia-dependent CPSase activities comparable to the wild-type protein. In contrast, the 27-kDa A2 and B2 recombinant proteins, which represent only 17% of the mass of the parent protein, were unable to use glutamine as a nitrogen donor, but the ammonia-dependent activity was enhanced 14–16-fold. The hyperactivity suggests that A2 and B2 are the catalytic subdomains and that A1 and B1 are attenuation domains which suppress the intrinsically high activity and are required for the physical association with the GLN subunit. Escherichia coli carbamoyl-phosphate synthetase (CPSase) is comprised of a 40-kDa glutaminase (GLN) and a 120-kDa synthetase (CPS) subunit. The CPS subunit consists of two homologous domains, CPS.A and CPS.B, which catalyze the two different ATP-dependent partial reactions involved in carbamoyl phosphate synthesis. Sequence similarities and controlled proteolysis experiments suggest that the CPS subdomains consist, in turn, of three subdomains, designated A1, A2, A3 and B1, B2, B3 for CPS.A and CPS.B, respectively. Previous studies of individually cloned CPS.A and CPS.B from E. coli and mammalian CPSase have shown that homologous dimers of either of these “half-molecules” could catalyze all three reactions involved in ammonia-dependent carbamoyl phosphate synthesis. Four smaller recombinant proteins were made for this study as follows: 1) A1-A2 in which the A3 subdomain was deleted from CPS.A, 2) B1-B2 lacking subdomain B3 of CPS.B, 3) the A2 subdomain of CPS.A, and 4) the B2 subdomain of CPS.B. When associated with the GLN subunit, A1-A2 and B1-B2 had both glutamine- and ammonia-dependent CPSase activities comparable to the wild-type protein. In contrast, the 27-kDa A2 and B2 recombinant proteins, which represent only 17% of the mass of the parent protein, were unable to use glutamine as a nitrogen donor, but the ammonia-dependent activity was enhanced 14–16-fold. The hyperactivity suggests that A2 and B2 are the catalytic subdomains and that A1 and B1 are attenuation domains which suppress the intrinsically high activity and are required for the physical association with the GLN subunit. Carbamoyl-phosphate synthetase (CPSase, 1The abbreviations used are: CPSase, carbamoyl-phosphate synthetase or its activity; CAD, the multifunctional protein having glutamine-dependent carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase activities; CPS, the synthetase domain or subunit of carbamoyl-phosphate synthetase; GLN, the glutaminase domain or subunit of carbamoyl-phosphate synthetase; GLNase, glutaminase; kb, kilobase pair(s). EC 6.3.5.5) catalyzes the formation of carbamoyl phosphate from bicarbonate, NH3, usually derived from glutamine, and ATP (1Meister A. Adv. Enzymol. Relat. Areas Mol. Biol. 1989; 62: 315-374PubMed Google Scholar, 2Anderson P.M. Meister A. Biochemistry. 1965; 4: 2803-2809Crossref PubMed Scopus (111) Google Scholar). The structure of CPSases can be quite diverse. For example, the monofunctional Escherichia coli CPSase (3Trotta P.P. Burt M.E. Haschemeyer R.H. Meister A. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 2599-2603Crossref PubMed Scopus (102) Google Scholar, 4Trotta P.P. Pinkus L.M. Haschemeyer R.H. Meister A. J. Biol. Chem. 1974; 249: 492-499Abstract Full Text PDF PubMed Google Scholar) consists of a 40-kDa glutaminase (GLN) subunit and a 120-kDa synthetase (CPS) subunit, whereas in its mammalian counterpart (5Shoaf W.T. Jones M.E. Biochemistry. 1973; 12: 4039-4051Crossref PubMed Scopus (143) Google Scholar, 6Mori M. Ishida H. Tatibana M. Biochemistry. 1975; 14: 2622-2630Crossref PubMed Scopus (79) Google Scholar, 7Coleman P.F. Suttle D.P. Stark G.R. J. Biol. Chem. 1977; 252: 6379-6385Abstract Full Text PDF PubMed Google Scholar), the GLN and CPS domains are fused and are part of CAD, a multifunctional protein that also has aspartate transcarbamoylase and dihydroorotase activities. Despite the differences in structural organization, the amino acid sequences are clearly similar (8Nyunoya H. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4629-4633Crossref PubMed Scopus (123) Google Scholar, 9Lusty C.J. Widgren E.E. Broglie K.E. Nyunoya H. J. Biol. Chem. 1983; 258: 14466-14477Abstract Full Text PDF PubMed Google Scholar, 10Nyunoya H. Lusty C.J. J. Biol. Chem. 1984; 259: 9790-9798Abstract Full Text PDF PubMed Google Scholar, 11Nyunoya H. Broglie K.E. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2244-2246Crossref PubMed Scopus (36) Google Scholar, 12Freund J.N. Jarry B.P. J. Mol. Biol. 1987; 193: 1-13Crossref PubMed Scopus (85) Google Scholar, 13Souciet J.-L. Nagy M. Legouar M. Lacroute F. Potier S. Gene (Amst .). 1989; 79: 59-70Crossref PubMed Scopus (81) Google Scholar, 14Faure M. Carmonis J.H. Jacquet M. Eur. J. Biochem. 1989; 179: 345-358Crossref PubMed Scopus (63) Google Scholar, 15Simmer 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, 16Bein K. Simmer J.P. Evans D.R. J. Biol. Chem. 1991; 266: 3791-3799Abstract Full Text PDF PubMed Google Scholar, 17Quinn C.L. Stephenson B.T. Switzer R.L. J. Biol. Chem. 1991; 266: 9113-9127Abstract Full Text PDF PubMed Google Scholar, 18Ghim S.Y. Nielsen P. Neuhard J. Microbiology. 1994; 140: 479-491Crossref PubMed Scopus (32) Google Scholar, 19Hong J. Salo W.L. Lusty C.J. Anderson P.M. J. Mol. Biol. 1994; 243: 131-140Crossref PubMed Scopus (59) Google Scholar, 20Helbing C.C. Atkinson B.G. J. Biol. Chem. 1994; 269: 11743-11750Abstract Full Text PDF PubMed Google Scholar, 21Hong J. Salo W.L. Anderson P.M. J. Biol. Chem. 1995; 270: 14130-14139Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) suggesting that all of these molecules are comprised of homologous domains and subdomains with analogous functions. The isolated GLN subunit of E. coli CPSase (3Trotta P.P. Burt M.E. Haschemeyer R.H. Meister A. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 2599-2603Crossref PubMed Scopus (102) Google Scholar, 4Trotta P.P. Pinkus L.M. Haschemeyer R.H. Meister A. J. Biol. Chem. 1974; 249: 492-499Abstract Full Text PDF PubMed Google Scholar) and the separately cloned GLN domain of CAD (22Guy H.I. Evans D.R. J. Biol. Chem. 1994; 269: 7702-7708Abstract Full Text PDF PubMed Google Scholar) hydrolyze glutamine (Reaction 1) and transfer ammonia to the CPS domain.glutamine+H2O→glutamate+NH3Equation 1 HO⎻O∥C⎻O−+ATP→HOO∥C⎻O⎻O∥P‖O−⎻O−+ADPEquation 2 HO⎻O−∥C⎻O⎻O∥P‖O−⎻O−+NH3→NH2O∥C⎻O−+PiEquation 3 NH2⎻O∥C⎻O−+ATP→NH2O∥C⎻O⎻O∥P‖O−⎻O−+ADPEquation 4 All of the other partial reactions (Reactions 2–4) occur on the CPS domain or subunit (3Trotta P.P. Burt M.E. Haschemeyer R.H. Meister A. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 2599-2603Crossref PubMed Scopus (102) Google Scholar, 4Trotta P.P. Pinkus L.M. Haschemeyer R.H. Meister A. J. Biol. Chem. 1974; 249: 492-499Abstract Full Text PDF PubMed Google Scholar). The determination of the amino acid sequence of CPSase from many different organisms (8Nyunoya H. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4629-4633Crossref PubMed Scopus (123) Google Scholar, 9Lusty C.J. Widgren E.E. Broglie K.E. Nyunoya H. J. Biol. Chem. 1983; 258: 14466-14477Abstract Full Text PDF PubMed Google Scholar, 10Nyunoya H. Lusty C.J. J. Biol. Chem. 1984; 259: 9790-9798Abstract Full Text PDF PubMed Google Scholar, 11Nyunoya H. Broglie K.E. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2244-2246Crossref PubMed Scopus (36) Google Scholar, 12Freund J.N. Jarry B.P. J. Mol. Biol. 1987; 193: 1-13Crossref PubMed Scopus (85) Google Scholar, 13Souciet J.-L. Nagy M. Legouar M. Lacroute F. Potier S. Gene (Amst .). 1989; 79: 59-70Crossref PubMed Scopus (81) Google Scholar, 14Faure M. Carmonis J.H. Jacquet M. Eur. J. Biochem. 1989; 179: 345-358Crossref PubMed Scopus (63) Google Scholar, 15Simmer 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, 16Bein K. Simmer J.P. Evans D.R. J. Biol. Chem. 1991; 266: 3791-3799Abstract Full Text PDF PubMed Google Scholar, 17Quinn C.L. Stephenson B.T. Switzer R.L. J. Biol. Chem. 1991; 266: 9113-9127Abstract Full Text PDF PubMed Google Scholar, 18Ghim S.Y. Nielsen P. Neuhard J. Microbiology. 1994; 140: 479-491Crossref PubMed Scopus (32) Google Scholar, 19Hong J. Salo W.L. Lusty C.J. Anderson P.M. J. Mol. Biol. 1994; 243: 131-140Crossref PubMed Scopus (59) Google Scholar, 20Helbing C.C. Atkinson B.G. J. Biol. Chem. 1994; 269: 11743-11750Abstract Full Text PDF PubMed Google Scholar, 21Hong J. Salo W.L. Anderson P.M. J. Biol. Chem. 1995; 270: 14130-14139Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) revealed that the CPS domain of these molecules invariably consists of two highly homologous halves, designated CPS.A and CPS.B. The two ATP-dependent partial reactions (Reactions 2 and 4) occur at different sites, and there is now convincing evidence (23Post L.E. Post D.J. Raushel F.M. J. Biol. Chem. 1990; 265: 7742-7747Abstract Full Text PDF PubMed Google Scholar, 24Miles B.W. Mareya S.M. Post L.E. Post D.J. Chang S.H. Raushel F.M. Biochemistry. 1993; 32: 232-240Crossref PubMed Scopus (32) Google Scholar, 25Guillou F. Liao M. Garcia-Espana A. Lusty C.J. Biochemistry. 1992; 31: 1656-1664Crossref PubMed Scopus (35) Google Scholar, 26Lusty C.J. Liao M. Biochemistry. 1993; 32: 1278-1284Crossref PubMed Scopus (19) Google Scholar) that CPS.A catalyzes the activation of bicarbonate, whereas the phosphorylation of carbamate to form carbamoyl phosphate is catalyzed by CPS.B. The site of carbamate formation (Reaction 3) is not known, but the rate of production of NH3 and carboxy phosphate is precisely coordinated, and once formed, these intermediates probably react spontaneously. Although CPS.A and CPS.B have different specific functions when fused together in the wild-type protein, the isolated domains are functionally equivalent. When CPS.A and CPS.B were separately cloned (27Guy H.I. Evans D.R. J. Biol. Chem. 1996; 271: 13762-13769Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), the half-molecules could each catalyze both partial reactions and the overall synthesis of carbamoyl phosphate from ammonia. When the mammalian domains are fused to the GLN domain, glutamine is hydrolyzed and the ammonia thus produced is used for carbamoyl phosphate synthesis. Thus, the catalytic sites are located within both CPS.A and CPS.B. There is extensive evidence that CPS.A and CPS.B domains are in turn comprised of subdomains. The CPSase model (15Simmer 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, 28Guillou 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, 29Kim H. Kelly R.E. Evans D.R. J. Biol. Chem. 1992; 267: 7177-7184Abstract Full Text PDF PubMed Google Scholar), illustrated in Fig. 1, is based in part on sequence homology (8Nyunoya H. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4629-4633Crossref PubMed Scopus (123) Google Scholar, 15Simmer 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, 30Lim F. Morris C.P. Occhiodoro F. Wallace J.C. J. Biol. Chem. 1988; 263: 11493-11497Abstract Full Text PDF PubMed Google Scholar, 31Takai T. Yokoyama C. Wada K. Tanabe T. J. Biol. Chem. 1988; 263: 2651-2657Abstract Full Text PDF PubMed Google Scholar, 32Rubio V. Cervera J. Lusty C.J. Bendala E. Britton H.G. Biochemistry. 1991; 30: 1068-1075Crossref PubMed Scopus (59) Google Scholar) between regions of the carbamoyl-phosphate synthetase and several different kinases. Moreover, controlled proteolysis of CAD (29Kim H. Kelly R.E. Evans D.R. J. Biol. Chem. 1992; 267: 7177-7184Abstract Full Text PDF PubMed Google Scholar, 33Mally M.I. Grayson D.R. Evans D.R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6647-6651Crossref PubMed Scopus (46) Google Scholar, 34Rumsby P.C. Campbell P.C. Niswander L.A. Davidson J.N. Biochem. J. 1984; 217: 435-440Crossref PubMed Scopus (15) Google Scholar, 35Grayson D.R. Lee L. Evans D.R. J. Biol. Chem. 1985; 260: 15840-15849Abstract Full Text PDF PubMed Google Scholar), the mammalian urea cycle enzyme, CPSase I (36Powers-Lee S.G. Corina K. J. Biol. Chem. 1986; 261: 15349-15352Abstract Full Text PDF PubMed Google Scholar, 37Guadalajara A. Grisolia S. Rubio V. Eur. J. Biochem. 1987; 165: 163-169Crossref PubMed Scopus (13) Google Scholar, 38Evans D.R. Balon M.A. Biochim. Biophys. Acta. 1988; 953: 185-196Crossref PubMed Scopus (25) Google Scholar), and more recentlyE. coli CPSase (32Rubio V. Cervera J. Lusty C.J. Bendala E. Britton H.G. Biochemistry. 1991; 30: 1068-1075Crossref PubMed Scopus (59) Google Scholar, 39Mareya S.M. Raushel F.M. Bioorg. Med. Chem. 1995; 3: 525-532Crossref PubMed Scopus (8) Google Scholar) showed that cleavage occurs at or near the junctions of many of the putative subdomains. The regions designated A2 and B2 have been implicated in ATP binding. Consensus sequences for the nucleotide binding sites (8Nyunoya H. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4629-4633Crossref PubMed Scopus (123) Google Scholar, 40Powers-Lee S.G. Corina K. J. Biol. Chem. 1987; 262: 9052-9056Abstract Full Text PDF PubMed Google Scholar) and the active site of the homologous kinases (8Nyunoya H. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4629-4633Crossref PubMed Scopus (123) Google Scholar, 15Simmer 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, 30Lim F. Morris C.P. Occhiodoro F. Wallace J.C. J. Biol. Chem. 1988; 263: 11493-11497Abstract Full Text PDF PubMed Google Scholar, 31Takai T. Yokoyama C. Wada K. Tanabe T. J. Biol. Chem. 1988; 263: 2651-2657Abstract Full Text PDF PubMed Google Scholar, 32Rubio V. Cervera J. Lusty C.J. Bendala E. Britton H.G. Biochemistry. 1991; 30: 1068-1075Crossref PubMed Scopus (59) Google Scholar) are located in the central region of CPS.A and CPS.B. Chemical modification of CAD (41Kim H.S. Lee L. Evans D.R. Biochemistry. 1991; 30: 10322-10329Crossref PubMed Scopus (21) Google Scholar) and mammalian CPSase I (40Powers-Lee S.G. Corina K. J. Biol. Chem. 1987; 262: 9052-9056Abstract Full Text PDF PubMed Google Scholar, 42Alonso E. Cervera J. Garcia-Espana A. Bendala E. Rubio V. J. Biol. Chem. 1992; 267: 4524-4532Abstract Full Text PDF PubMed Google Scholar, 43Potter M.D. Powers-Lee S.G. J. Biol. Chem. 1992; 267: 2023-2031Abstract Full Text PDF PubMed Google Scholar, 44Potter M.D. Powers-Lee S.G. Arch. Biochem. Biophys. 1993; 306: 377-382Crossref PubMed Scopus (9) Google Scholar, 45Alonso E. Rubio V. Eur. J. Biochem. 1995; 229: 377-384Crossref PubMed Scopus (21) Google Scholar) as well as site-directed mutagenesis of E. coli CPSase (23Post L.E. Post D.J. Raushel F.M. J. Biol. Chem. 1990; 265: 7742-7747Abstract Full Text PDF PubMed Google Scholar, 46Stapleton 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, 47Javid-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) showed that ATP binds to A2 and B2. The conclusions of the biochemical studies are consistent with the recently solved x-ray structure (65) of E. coli carbamoyl-phosphate synthetase. To establish whether the subdomains of CPS.A and CPS.B correspond to autonomously folded substructural elements with specific functions, we have separately cloned and expressed some of the individual E. coli CPSase subdomains and combinations of subdomains. The 7.9-kb plasmid, pLLK12 encoding both subunits of E. coli CPSase, and the 6.8-kb plasmid, pHN12, which encodes the E. coli CPSase large subunit, were kindly provided by Dr. Carol Lusty (The Public Health Research Institute of the City of New York) as were the E. colistrains, RC50 and L673 (28Guillou 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) which are each defective in both thecarA and carB genes, encoding the E. coli CPSase GLN and CPS subunits, respectively. The strain, L673, is also defective in the Lon protease. The plasmid, pABGB6, encodes theE. coli GLN and CPSase B subdomain of E. coliCPSase. 2A. Bouvier, H. Guy, and D. R. Evans, unpublished results. The recombinant proteins, which are expressed constitutively under the control of thecarAB promoter, were isolated from 60-ml cultures of transformed L673 cells grown to stationary phase (28Guillou 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 3 ml of 50 mm HEPES, 10% glycerol, pH 7.0, and broken by sonication for 2 min at 4 °C. The sonicate was centrifuged at 29,000 × g for 30 min at 4 °C. Transformation was carried out by the Hanahan procedure (48Hanahan D. Glover D.M. DNA Cloning: A Practical Approach. IRL Press at Oxford University Press, Oxford1985Google Scholar). Restriction digests, ligations, and other DNA methods were carried out using standard protocols (49Maniatis T. Fritsch E.R. Sambrook I. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). The plasmid, pHGGA12, encoding the E. coli GLN and CPS A1-A2 subdomains (Fig. 1, residues 1–359), was constructed by reacting the plasmid, pLLK12, which encodes the wild-type CPS, with NspV (2730) and SmaI (site in the vector). The 5.4-kb fragment was then religated using T4 DNA ligase following treatment with the Klenow fragment of DNA polymerase. The expressed protein includes an additional 4 residues from A3 and 7 residues from the vector appended to the carboxyl end of A1-A2. The plasmid, pHGGA12, was then reacted with BssHII-(1932) and ApaI-(1688) and the ends made flush using mung bean nuclease prior to ligation. The resulting 5.2-kb plasmid, pHGGA2, encodes the E. coli GLN and CPSA2-(111–359) subdomain and 15 amino acid residues derived from the amino end of A1. The carboxyl end of the protein is the same as A1-A2 described above. The construct encoding B1-B2 (residues 560–892) was derived from the 6.4-kb plasmid, pABGB6, which encodes the E. coli GLN subunit, the CPS.B subdomains, and part of the carboxyl end of CPS.A. The plasmid was reacted with AvaI-(4339) and SstI (in the vector) and the ends were made flush using the Klenow fragment of DNA polymerase and then religated. The resulting 5.6-kb plasmid, pHGB12, encodes the GLN subunit and CPSB12 and included an additional 53 residues derived from A3 and 7 residues from B3 on the amino and carboxyl ends, respectively. The plasmid, pHN12, encoding the large subunit of E. coliCPS, was reacted with ApaI-(1688) andPinAI-(3616), and the resulting 4.4-kb fragment encodingE. coli B2 and B3 was reacted with the Klenow fragment and T4 DNA ligase to yield pHGB2B3. The plasmid, pHGB2, encoding only the B2 subdomain (residue 658–892), was constructed by reacting pHGB2B with AvaI-(4339) and SstI (in the vector). The 3.5-kb fragment was then reacted with the Klenow fragment and religated. The recombinant protein has the 7 amino acid residues of the amino end of B3 and 4 residues from the vector appended to the carboxyl end. All of the plasmids, except pHGB2, also contain the carA gene so that the GLN subunit is also expressed as a separate polypeptide. The four recombinant plasmids constructed for this study also encoded the first 15 amino acids of the E. coli CPS subunit which are appended to the amino end of the expressed protein. It is very unlikely that the presence of these residues alters the function of the recombinant proteins, since the amino end of the CPSase synthetase domain is poorly conserved among members of this family of proteins, both in sequence and in the number of residues preceding the core A1 domain. Moreover 12 of these same 15 residues (50Guy H.I. Rotgeri A. Evans D.R. J. Biol. Chem. 1997; 272: 19913-19918Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) were deleted from the amino end of the CPS.A domain without effect on the properties of the E. coli enzyme. Nevertheless, we are now refining these constructs to remove these additional residues and more precisely define the limits of the functional domains. The procedures for the isolation of the wild type E. coli CPSase (28Guillou 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) and the CPS subunit (51Rubino S.D. Nyunoya H. Lusty C.J. J. Biol. Chem. 1987; 262: 4382-4386Abstract Full Text PDF PubMed Google Scholar) from pLLK12 and pHN12, respectively, were described previously. Protein concentrations were determined by the Bradford dye-binding method (52Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217508) Google Scholar) and by scanning Coomassie Blue-stained polyacrylamide gels. SDS-gel electrophoresis was carried out on 7.5–15% linear polyacrylamide gradients (53Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207523) Google Scholar). The gels were scanned, and the concentration of the individual 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. Carbamoyl-phosphate synthetase activity was assayed using a radiometric procedure (7Coleman P.F. Suttle D.P. Stark G.R. J. Biol. Chem. 1977; 252: 6379-6385Abstract Full Text PDF PubMed Google Scholar, 33Mally M.I. Grayson D.R. Evans D.R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6647-6651Crossref PubMed Scopus (46) Google Scholar), whereas the GLNase activity was measured (23Post L.E. Post D.J. Raushel F.M. J. Biol. Chem. 1990; 265: 7742-7747Abstract Full Text PDF PubMed Google Scholar) spectrophotometrically. The only modification was that the GLNase assay buffer also contained 15% Me2SO and 2.5% glycerol. The molecular mass was determined by gel filtration chromatography using an open 1.9 × 42-cm Sephacryl S-300 column equilibrated with 0.5 mTris-HCl, pH 7.4, 1 mm dithiothreitol, and 5% glycerol. Alternatively, a Pharmacia FPLC system fitted with a 1.6 × 50-cm Sephacryl S-300 high resolution (HR 16/50) Pharmacia column was used. The column was equilibrated in 0.1 mKH2PO4, 1 mm EDTA, pH 7.5, and eluted at a flow rate of 0.4 ml/min at room temperature. Column fractions were analyzed by measuring the absorbance at 280 nm, SDS-gel electrophoresis, and CPSase assays. The column was calibrated with carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), and blue dextran. Extensive sequence identity (15Simmer 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) suggests that E. coli and mammalian CPSases have common subdomain structures. We elected to work with E. coli CPSase, rather than the mammalian enzyme, because the level of expression of the bacterial protein in E. coli tends to be higher. The assignment of theE. coli CPS subdomain junctions, shown in Fig.1, was based on the CAD substructural analysis (29Kim H. Kelly R.E. Evans D.R. J. Biol. Chem. 1992; 267: 7177-7184Abstract Full Text PDF PubMed Google Scholar) by aligning the sequences of the mammalian and bacterial proteins. The construction (Fig. 2) of the four recombinant plasmids used in this study is described under “Experimental Procedures.” Plasmid pHGGA12 encodes the fused A1-A2 subdomains; pHGGB12 encodes B1-B2, and pHGGA2 encodes the A2 subdomain. These three plasmids also contain the intactcarA gene so that the GLN subunit is also expressed as a separate polypeptide chain. The plasmid pHGB2, encoding the B2 subdomain, does not include the carA gene. In L673 cells transformed with pHGGA12 and pHGGB12, the 42-kDa GLN subunit was expressed along with the 40- and a 44-kDa fragment, respectively. A1-A2 and B1-B2 were produced (Fig.3) as soluble, stable proteins representing 21 and 14% of the total cellular protein, respectively. Transformation of the same strain with pHGGA2 and pHGB2 produced much lower levels, 0.4% of the soluble 31-kDa A2 and 28-kDa B2 subdomains. The function of the recombinant proteins was assayed in cell extracts prepared as described under “Experimental Procedures.” The A1-A2 and B1-B2 subdomains were both found to catalyze ammonia-dependent carbamoyl phosphate synthesis. The ATP saturation curves (Fig.4 A) of A1-A2 exhibit typical Michaelis-Menten kinetics with a K m for ATP (TableI) that was about 3-fold higher than the value obtained for native E. coli CPSase. Thek cat2 values of A1-A2 are approximately the same as that of the parent molecule. For B1-B2 (Fig.4 B and Table I), the k cat values are lower. This result is probably due to appreciable substrate inhibition exhibited by B1-B2, which made it difficult to obtain accurate values for the kinetic parameters.Table ISummary of kinetic parameters from ATP saturation curvesProteinGln-CPSaseNH3-CPSaseK mV maxk cat1-aCalculated from the V max assuming a molecular mass of 120 kDa for E. coli CPSase synthetase subunit, and 40, 44, 31, and 28 kDa for A1-A2, B1-B2, A2 and B2, respectively. For these calculations it was assumed that two copies of the CPS subdomains interact to produce carbamoyl phosphate (see “Discussion”), one catalyzes the ATP dependent activation of bicarbonate and the other catalyzes the phosphorylation of carbamate.K mV maxk cat1-aCalculated from the V max assuming a molecular mass of 120 kDa for E. coli CPSase synthetase subunit, and 40, 44, 31, and 28 kDa for A1-A2, B1-B2, A2 and B2, respectively. For these calculations it was assumed that two copies of the CPS subdomains interact to produce carbamoyl phosphate (see “Discussion”), one catalyzes the ATP dependent activation of bicarbonate and the other catalyzes the phosphorylation of carbamate.mmμmol/min/mgs −1mmμmol/min/mgs −1CPSase0.43 ± 0.023.00 ± 0.106.00 ± 0.540.32 ± 0.032.61 ± 0.135.22 ± 0.70A1-A21.25 ± 0.214.02 ± 0.315.36 ± 0.410.82 ± 0.064.15 ± 0.135.53 ± 0.23B1-B20.151-bThe saturation curves exhibited inhibition at high ATP concentrations and thus did not conform to the Michaelis-Menten equation. The values given for the K m correspond to the concentration of ATP at half of the maximum observed activity, andV max corresponds to the maximum observed activity.2.241-bThe saturation curves exhibited inhibition at high ATP concentrations and thus did not conform to the Michaelis-Menten equation. The values given for the K m correspond to the concentration of ATP at half of the maximum observed activity, andV max corresponds to the maximum observed activity.3.290.381-bThe saturation curves exhibited inhibition at high ATP concentrations and thus did not conform to the Michaelis-Menten equation. The values given for the K m correspond to the concentration of ATP at half of the maximum observed activity, andV max corresponds to the maximum observed activity. 1.291-bThe saturation curves exhibited inhibition at high ATP concentrations and thus did not conform to the Michaelis-Menten equation. The values given for the K m correspond to the concentration of ATP at half of the maximum observed activity, andV max corresponds to the maximum observed activity. 1.89A2ND1-cNot determined.000.361-bThe saturation curves exhibited inhibition at high ATP concentrations and thus did not conform to the Michaelis-Menten equation. The values given for the K m correspond to the concentration of ATP at half of the maximum observed activity, andV max corresponds to the maximum observed activity.72.61-bThe saturation curves exhibited inhibition at high ATP concentrations and thus did not conform to the Michaelis-Menten equation. The values given for the K m correspond to the concentration of ATP at half of the maximum observed activity, andV max corresponds to the maximum observed activity.75.0B2ND1-cNot determined.000.62 ± 0.0891.9 ± 4.9485.8 ± 2.3These parameters were obtained by least squares fit to the Michaelis-Menten equation.1-a Calculated from the V max assuming a molecular mass of 120 kDa for E. coli CPSase synthetase subunit, and 40, 44, 31, and 28 kDa for A1-A2, B1-B2, A2 and B2, respectively. For these calculations it was assumed that two copies of the CPS subdomains interact to produce carbamoyl phosphate (see “Discussion”), one catalyze

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