Key points are not available for this paper at this time.
Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that also catalyzes the second and third steps of pyrimidine biosynthesis. Carbamoyl phosphate synthesis requires the concerted action of the glutaminase (GLN) and carbamoyl-phosphate synthetase domains of CAD. There is a functional linkage between these domains such that glutamine hydrolysis on the GLN domain does not occur at a significant rate unless ATP and HCO3−, the other substrates needed for carbamoyl phosphate synthesis, bind to the synthetase domain. The GLN domain consists of catalytic and attenuation subdomains. In the separately cloned GLN domain, the catalytic subdomain is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage. Replacement of Ser44 in the GLN attenuation domain with alanine increases thekcat/Km for glutamine hydrolysis 680-fold. The formation of a functional hybrid between the mammalian Ser44 GLN domain and the Escherichia coli carbamoyl-phosphate synthetase large subunit had little effect on glutamine hydrolysis. In contrast, ATP and HCO3− did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted. In accord with this interpretation, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis were no longer coordinated. Approximately 3 times more glutamine was hydrolyzed by the Ser44 → Ala mutant than that needed for carbamoyl phosphate synthesis. Ser44, the only attenuation subdomain residue that extends into the GLN active site, appears to be an integral component of the regulatory circuit that phases glutamine hydrolysis and carbamoyl phosphate synthesis. Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that also catalyzes the second and third steps of pyrimidine biosynthesis. Carbamoyl phosphate synthesis requires the concerted action of the glutaminase (GLN) and carbamoyl-phosphate synthetase domains of CAD. There is a functional linkage between these domains such that glutamine hydrolysis on the GLN domain does not occur at a significant rate unless ATP and HCO3−, the other substrates needed for carbamoyl phosphate synthesis, bind to the synthetase domain. The GLN domain consists of catalytic and attenuation subdomains. In the separately cloned GLN domain, the catalytic subdomain is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage. Replacement of Ser44 in the GLN attenuation domain with alanine increases thekcat/Km for glutamine hydrolysis 680-fold. The formation of a functional hybrid between the mammalian Ser44 GLN domain and the Escherichia coli carbamoyl-phosphate synthetase large subunit had little effect on glutamine hydrolysis. In contrast, ATP and HCO3− did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted. In accord with this interpretation, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis were no longer coordinated. Approximately 3 times more glutamine was hydrolyzed by the Ser44 → Ala mutant than that needed for carbamoyl phosphate synthesis. Ser44, the only attenuation subdomain residue that extends into the GLN active site, appears to be an integral component of the regulatory circuit that phases glutamine hydrolysis and carbamoyl phosphate synthesis. In mammals and most other species, the synthesis of carbamoyl phosphate in the de novo pyrimidine biosynthetic pathway occurs in a series of four partial reactions (1Anderson P.M. Meister A. Biochemistry. 1966; 5: 3157-3163Crossref PubMed Scopus (93) Google Scholar, 2Meister A. Adv. Enzymol. Relat. Areas Mol. Biol. 1989; 62: 315-374PubMed Google Scholar) that are catalyzed by distinct structural domains of carbamoyl-phosphate synthetase (CPSase, 1The abbreviations used are:CPSasecarbamoyl-phosphate synthetase activityCADthe multifunctional protein having glutamine-dependent carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase activitiesCPSthe synthetase domain or subunit of carbamoyl-phosphate synthetaseGLNthe amidotransferase or glutaminase domain or subunit of carbamoyl-phosphate synthetaseGLNaseglutaminase activityGLN-CPSthe hybrid CPSase consisting of the mammalian GLN domain and the E. coli CPS domainPCRpolymerase chain reactionHPLChigh performance liquid chromatographyOPAo-phthaldialdehyde EC 6.3.5.5). Glutamine+H2O→glutamate+NH3REACTION 1 ATP+HCO3−→carboxy phosphate+ADPREACTION 2 Carboxy phosphate+NH3→carbamate+PiREACTION 3 ATP+carbamate→carbamoyl phosphate+ADPREACTION 4 Mammalian carbamoyl-phosphate synthetase is part of a large multifunctional protein called CAD (3Shoaf W.T. Jones M.E. Biochemistry. 1973; 12: 4039-4051Crossref PubMed Scopus (143) Google Scholar, 4Mori M. Ishida H. Tatibana M. Biochemistry. 1975; 14: 2622-2630Crossref PubMed Scopus (79) Google Scholar, 5Coleman P. Suttle D. Stark G. J. Biol. Chem. 1977; 252: 6379-6385Abstract Full Text PDF PubMed Google Scholar), which also has aspartate transcarbamoylase and dihydroorotase activities, enzymes that catalyze the second and third steps of the de novo pathway, respectively. The 243-kDa CAD polypeptide is organized into discrete structural domains each with a specific function (6Mally M.I. Grayson D.R. Evans D.R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6647-6651Crossref PubMed Scopus (46) Google Scholar, 7Davidson J.N. Rumsby P.C. Tamaren J. J. Biol. Chem. 1981; 256: 5220-5225Abstract Full Text PDF PubMed Google Scholar, 8Grayson D.R. Lee L. Evans D.R. J. Biol. Chem. 1985; 260: 15840-15849Abstract Full Text PDF PubMed Google Scholar, 9Kim H. Kelly R.E. Evans D.R. J. Biol. Chem. 1992; 267: 7177-7184Abstract Full Text PDF PubMed Google Scholar). The 38-kDa glutaminase (GLN) domain located on the amino end of the polypeptide (10Simmer 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, 11Bein K. Simmer J. Evans D. J. Biol. Chem. 1991; 266: 3791-3799Abstract Full Text PDF PubMed Google Scholar) catalyzes the hydrolysis of glutamine and transfers the ammonia to the adjacent 120-kDa synthetase (CPS) domain, where the remaining partial reactions take place. The CPS domain of CAD (10Simmer 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) and all known CPSases (12Nyunoya H. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4629-4633Crossref PubMed Scopus (123) Google Scholar, 13Anderson P.M. Walsh P.J. Wright P.A. Nitrogen Metabolism and Excretion. CRC Press, Boca Raton, FL1995: 33-55Google Scholar) consist of two homologous subdomains, CPS.A and CPS.B (10Simmer 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), that probably arose as a result of an ancestral gene duplication and fusion (12Nyunoya H. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4629-4633Crossref PubMed Scopus (123) Google Scholar). Escherichia coli CPSase is a monofunctional protein (14Trotta 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, 15Trotta P.P. Estis L.F. Meister A. Haschemeyer R.H. J. Biol. Chem. 1974; 249: 482-489Abstract Full Text PDF PubMed Google Scholar) consisting of a 42-kDa GLN subunit and a 120-kDa CPS subunit. Despite differences in structural organization, the sequence and domain structure of the mammalian and bacterial proteins are very similar. There is extensive evidence that the two different ATP-dependent partial reactions, the activation of bicarbonate (Reaction 2) and the phosphorylation of carbamate (Reaction 4), are catalyzed by CPS.A and CPS.B, respectively (16Powers-Lee S.G. Mastico R.A. Bendayan M. J. Biol. Chem. 1987; 262: 15683-15688Abstract Full Text PDF PubMed Google Scholar, 17Boettcher B.R. Meister A. J. Biol. Chem. 1980; 255: 7129-7133Abstract Full Text PDF PubMed Google Scholar, 18Kim H.S. Lee L. Evans D.R. Biochemistry. 1991; 30: 10322-10329Crossref PubMed Scopus (21) Google Scholar, 19Potter M.D. Powers-Lee S.G. J. Biol. 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Biochemistry. 1996; 35: 14352-14361Crossref PubMed Scopus (55) Google Scholar). carbamoyl-phosphate synthetase activity the multifunctional protein having glutamine-dependent carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase activities the synthetase domain or subunit of carbamoyl-phosphate synthetase the amidotransferase or glutaminase domain or subunit of carbamoyl-phosphate synthetase glutaminase activity the hybrid CPSase consisting of the mammalian GLN domain and the E. coli CPS domain polymerase chain reaction high performance liquid chromatography o-phthaldialdehyde The x-ray structure of the E. coli enzyme (23Thoden J.B. Holden H.M. Wesenberg G. Raushel F.M. Rayment I. Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (306) Google Scholar, 24Thoden J.B. Miran S.G. Phillips J.C. Howard A.J. Raushel F.M. Holden H.M. Biochemistry. 1998; 37: 8825-8831Crossref PubMed Scopus (89) Google Scholar, 25Thoden J.B. Wesenberg G. Raushel F.M. Holden H.M. Biochemistry. 1999; 38: 2347-2357Crossref PubMed Scopus (64) Google Scholar, 26Thoden J.B. Raushel F.M. Benning M.M. Rayment I. Holden H.M. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 8-24Crossref PubMed Scopus (86) Google Scholar) has been solved to a resolution of 1.8 Å. Remarkably, the active sites were found to be widely separated but connected by a narrow tunnel that passes through the interior of the molecule. The ammonia generated by hydrolysis of glutamine presumably diffuses through the tunnel to the active site of CPS.A, where it reacts with carboxy phosphate to form carbamate. The carbamate then diffuses through the tunnel to the active site of CPS.B, where carbamoyl phosphate is formed in the second ATP-dependent reaction. The mechanism of glutamine hydrolysis by CPSase (27Wellner V.P. Anderson P.M. Meister A. Biochemistry. 1973; 12: 2061-2066Crossref PubMed Scopus (24) Google Scholar, 28Chaparian M.G. Evans D.R. J. Biol. Chem. 1991; 266: 3387-3395Abstract Full Text PDF PubMed Google Scholar, 29Lusty C.J. Liao M. Biochemistry. 1993; 32: 1278-1284Crossref PubMed Scopus (19) Google Scholar, 30Miles B.W. Banzon J.A. Raushel F.M. Biochemistry. 1998; 37: 16773-16779Crossref PubMed Scopus (34) Google Scholar, 31Hewagama A. Guy H.I. Chaparian M. Evans D.R. Biochim. Biophys. Acta. 1998; 1388: 489-499Crossref PubMed Scopus (13) Google Scholar) and other trpG-type amidotransferases (32Mei B. Zalkin H. J. Biol. Chem. 1989; 264: 16613-16619Abstract Full Text PDF PubMed Google Scholar, 33Zalkin H. Smith J.L. Enzymes Utilizing Glutamine as an Amide Donor: in Amino Acid Metabolism, Part A. Vol. 72. John Wiley and Sons, Inc., New York1998: Scholar, F. Mol. Sci. 1998; PubMed Scopus Google Scholar) is to that of the The reaction through a and is evidence that a catalytic consisting of and in CAD The was in the x-ray structure J.B. Miran S.G. Phillips J.C. Howard A.J. Raushel F.M. Holden H.M. Biochemistry. 1998; 37: 8825-8831Crossref PubMed Scopus (89) Google Scholar) of an E. coli CPSase The of the catalytic the to the and in in was found to be to the of the The that this residue in by the for by the active site and by the in the The partial reactions are by a linkage between glutamine hydrolysis and carbamoyl phosphate synthesis. the GLN and CPS domains of E. coli and mammalian CPSase function (1Anderson P.M. Meister A. Biochemistry. 1966; 5: 3157-3163Crossref PubMed Scopus (93) Google Scholar, V.P. Anderson P.M. Meister A. Biochemistry. 1973; 12: 2061-2066Crossref PubMed Scopus (24) Google Scholar, 28Chaparian M.G. Evans D.R. J. Biol. Chem. 1991; 266: 3387-3395Abstract Full Text PDF PubMed Google Scholar, 29Lusty C.J. Liao M. Biochemistry. 1993; 32: 1278-1284Crossref PubMed Scopus (19) Google Scholar, 30Miles B.W. Banzon J.A. Raushel F.M. Biochemistry. 1998; 37: 16773-16779Crossref PubMed Scopus (34) Google Scholar, 31Hewagama A. Guy H.I. Chaparian M. Evans D.R. Biochim. Biophys. Acta. 1998; 1388: 489-499Crossref PubMed Scopus (13) Google Scholar, E. Anderson P.M. Meister A. Biochemistry. 1966; 5: PubMed Scopus Google Scholar, P.M. Meister A. Biophys. 1973; 55: PubMed Scopus Google Scholar, P.P. Haschemeyer R.H. Meister A. J. Biol. Chem. 1974; 249: Full Text PDF PubMed Google Scholar, P.M. Biochemistry. 1975; 14: PubMed Scopus Google Scholar) a functional linkage between the active sites that Glutamine hydrolysis does not at a significant rate in the of ATP and functional linkage the hydrolysis of glutamine the other substrates needed for carbamoyl phosphate synthesis are cloned and the mammalian GLN domain H.I. Evans D.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and that the protein a with the E. coli CPS subunit. The hybrid has that are to of the GLN domain of and the linkage between the GLN and CPS domains is functional A. Guy H.I. Chaparian M. Evans D.R. Biochim. Biophys. Acta. 1998; 1388: 489-499Crossref PubMed Scopus (13) Google Scholar, H.I. Evans D.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). used this hybrid to the of Ser44 and that it is not a catalytic residue in the but is a in the functional linkage that the reactions on the GLN and CPS was was and and and all other were The H.I. Evans D.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar) a the mammalian CAD GLN domain in a J. Biol. Chem. 1985; 260: Full Text PDF PubMed Google Scholar). The of the protein is of the The E. J. Biol. Chem. 1985; 260: Full Text PDF PubMed Google Scholar), the and and has a The high F. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1989; PubMed Scopus Google Scholar), which the gene that the large subunit of E. coli was by Lusty of the of New New as was the E. coli which is in the and E. coli carbamoyl-phosphate synthetase as as the the were a in with The of the mammalian GLN domain in was as H.I. Evans D.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the E. coli CPS subunit is H. Lusty C.J. J. Biol. Chem. 1987; 262: Full Text PDF PubMed Google Scholar) in with a the E. coli CPSase large subunit. were the and of E. coli were by the D. Vol. Press, New Scholar). was by the and which with high were the was with to the The was then in E. coli were and the and by the at the The mutant was then into the and other were I. Scholar). CAD was an of as P. Suttle D. Stark G. J. Biol. Chem. 1977; 252: 6379-6385Abstract Full Text PDF PubMed Google M.I. Grayson D.R. Evans D.R. J. Biol. Chem. 1980; 255: Full Text PDF PubMed Google Scholar). The E. coli CPSase large subunit was the of H. Lusty C.J. J. Biol. Chem. 1987; 262: Full Text PDF PubMed Google Scholar). The CAD GLN domain and the mutant were by the H.I. Evans D.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). form the hybrid CPSase of the CAD GLN domain or mutant and the E. coli CPSase large subunit were and for A. Guy H.I. Chaparian M. Evans D.R. Biochim. Biophys. Acta. 1998; 1388: 489-499Crossref PubMed Scopus (13) Google Scholar, H.I. Evans D.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The was then a at or by at 4 were by the as a M.M. PubMed Scopus Google Scholar). was on PubMed Scopus Google Scholar). The structure of the CAD domains was with E. coli CPSase as the the The of CAD and E. coli CPSase was to structure and sequence were in two of by of to The CPSase activity was at a (6Mally M.I. Grayson D.R. Evans D.R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6647-6651Crossref PubMed Scopus (46) Google Scholar, Jones M.E. J. Biol. Chem. 1980; 255: Full Text PDF PubMed Google Scholar) a 2 of the of ATP in the The activity A. Guy H.I. Chaparian M. Evans D.R. Biochim. Biophys. Acta. 1998; 1388: 489-499Crossref PubMed Scopus (13) Google Scholar) of the CAD GLN domain and the coli CPSase hybrid was by an that the glutamine the by as The or and glutamine in a of The reaction was by of the GLN domain in to for 1 at and then with of The on for to for at to the The was then with of glutamine than the were by of to the glutamine of was to of the protein the of a was the The activity is as of the GLN domain, and the in the of the GLN domain. The formation of a between the 38-kDa mammalian GLN domain and the with the coli CPSase synthetase was by The hybrid in 1 and was to a The was and at with the were by CPSase and by The mammalian GLN domain was the E. coli CPSase structure (23Thoden J.B. Holden H.M. Wesenberg G. Raushel F.M. Rayment I. Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (306) Google Scholar) as a the sequence of the mammalian and bacterial the of active site that for the E. coli Ser44 to in the E. coli is located the catalytic site of the GLN domain. The chain extends into the site with of the and the of the catalytic Ser44 was with alanine by the H.I. Evans D.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar) as a of the 38-kDa and mutant domains were the were into the E. coli proteins were to as H.I. Evans D.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar) on a as in the of the a of the mammalian Ser44 → Ala GLN domain and the E. coli CPSase CPS subunit formed a hybrid The GLN domain had very catalytic activity a of a and a very The formation of a hybrid consisting of the mammalian GLN domain and the E. coli CPS subunit the function of the GLN domain. The to the domain. The of the hybrid are to for for the glutaminase Chaparian and Evans Chaparian and Evans → Ala Chaparian and Evans M.G. Evans D.R. J. Biol. Chem. 1991; 266: 3387-3395Abstract Full Text PDF PubMed Google Scholar). in a to the that be a catalytic the of catalytic activity of the domain was by with alanine 2 and with the domain, the was the the the GLN domain, the by a of The formation of the hybrid with the Ser44 → Ala GLN domain a with to for the the are with the Ser44 → Ala GLN domain, for glutamine was in the mutant but was no Ser44 does not in glutamine hydrolysis in the domain or the hybrid in the of ATP and The Ser44 → Ala hybrid protein also catalyze the synthesis of carbamoyl for the reaction and and that the glutamine and ATP are very to the for the glutamine and ATP are for the mutant for carbamoyl phosphate ATP are and were to the these The is and for the and mutant respectively. The glutamine were to the ATP are and were to the these The is and for the and mutant respectively. The glutamine were to the The ATP are and were to the these The is and for the and mutant respectively. The glutamine were to the in a The of ATP and bicarbonate to the CPS subunit the activity of the hybrid and CAD and respectively. The is the result of a in significant in the for In the of ATP and bicarbonate for glutamine hydrolysis of the mutant hybrid is only with that of the hybrid and it does not in the of of these substrates the functional linkage that the reactions on the GLN and CPS domains is as a result of of Ser44 with In CAD and in the hybrid the rate of glutamine hydrolysis is to the rate of carbamoyl phosphate synthesis. The for glutamine hydrolysis in the of ATP and bicarbonate the rate of carbamoyl phosphate synthesis in the a 3 of the of carbamoyl phosphate 3 hydrolyzed at of glutamine a of in accord with the of the reaction at all of the In contrast, the rate of glutamine hydrolysis the rate of carbamoyl phosphate in the mutant for the reaction at ATP and bicarbonate is for the CPSase reaction is only the of glutamine carbamoyl phosphate had a of indicating that the for the reaction catalyzed by the enzyme is not in the result be Ser44 a in the reactions on the two Carbamoyl phosphate synthesis the concerted action of two domains that in are in the mechanism of glutamine hydrolysis and the interactions between the GLN and CPS domains of the mammalian multifunctional protein CAD. each of the functional as of CPS needed for the of and A. Guy H.I. Chaparian M. Evans D.R. Biochim. Biophys. Acta. 1998; 1388: 489-499Crossref PubMed Scopus (13) Google Scholar, H.I. Evans D.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar) that the hybrid consisting of the mammalian GLN domain and the E. coli CPS subunit has catalytic to CAD as as a functional interdomain linkage. used this to the of Ser44 in carbamoyl phosphate synthesis. the hydrolysis of glutamine by CAD be the GLN domain has activity as a result of an in the for glutamine and in the are to the found in CAD a is formed by the of the GLN domain and the CPS subunit of E. coli (10Simmer 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, 11Bein K. Simmer J. Evans D. J. Biol. Chem. 1991; 266: 3791-3799Abstract Full Text PDF PubMed Google Scholar) that the CPSase GLN domain consists of two distinct The of the domain is homologous to the amidotransferase domain of all or amidotransferases H. Smith J.L. Enzymes Utilizing Glutamine as an Amide Donor: in Amino Acid Metabolism, Part A. Vol. 72. John Wiley and Sons, Inc., New York1998: Scholar, F. Mol. Sci. 1998; PubMed Scopus Google Scholar, H. Enzymol. 1985; PubMed Scopus Google Scholar), the amino of the domain is to the amidotransferase domains of the other biosynthetic enzymes an of and not a chain to the amino of the CPS GLN domain, it was to that all of the for glutamine and be found in the of the CAD GLN domain. for this was cloned and H.I. Evans D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (13) Google Scholar) the two of the mammalian CPSase GLN domain and that are with specific The amino has no catalytic activity but a with the CPS domain. The the catalytic glutamine with the and is more active than the glutaminase activity for in the of ATP and HCO3− that a function of the amino of the GLN domain, the attenuation was to the high catalytic activity of the catalytic subdomain and that this of the was in the interdomain between the GLN and CPS In accord with this the x-ray structure of E. (23Thoden J.B. Holden H.M. Wesenberg G. Raushel F.M. Rayment I. Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (306) Google Scholar) that the attenuation subdomain extensive with the CPS subunit in E. coli The x-ray structure also that Ser44 is the only residue in the attenuation subdomain that extends into the GLN active site, it a for in the functional linkage. The activation of the GLN domain that occurs with the CPSase subunit is by the of Ser44 with alanine in the domain. increases to with a of for the hybrid The high activity of the Ser44 → Ala GLN domain that this residue is not in The also to in the Ser44 → Ala domain, but is than that of the hybrid that this residue is for the Ser44 → Ala GLN domain with coli CPS the is the as the hybrid it that the of activity in the GLN domain to a large the result of the alanine is with the of activity is to be a of that form a with of the active site that with function in the domain with the CPS Ser44 is to be to located in the of E. coli CPSase (23Thoden J.B. Holden H.M. Wesenberg G. Raushel F.M. Rayment I. Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (306) Google Scholar). the residue has been in the little be the Ser44 → Ala domain with the CPS subunit. with this interpretation, the for glutamine hydrolysis by the Ser44 → Ala hybrid are to the for the hybrid that Ser44 is not for glutamine hydrolysis by the hybrid in the of ATP and The hydrolysis of glutamine and the activation of bicarbonate are reactions that occur in to the hydrolysis of glutamine or ATP that is the active sites are in the E. coli structure (23Thoden J.B. Holden H.M. Wesenberg G. Raushel F.M. Rayment I. Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (306) Google Scholar), and ammonia be to the active site of CPS.A a interdomain The of these partial reactions requires that the activity be that it is not at or catalytic unless the of the other substrates needed for carbamoyl phosphate synthesis are part of the interdomain functional linkage is the of the glutaminase activity ATP and bicarbonate are and of CAD M.G. Evans D.R. J. Biol. Chem. 1991; 266: 3387-3395Abstract Full Text PDF PubMed Google Scholar, 31Hewagama A. Guy H.I. Chaparian M. Evans D.R. Biochim. Biophys. Acta. 1998; 1388: 489-499Crossref PubMed Scopus (13) Google Scholar) in the of ATP and the and the rate for the of the is the as the for glutamine indicating that it is the ATP and bicarbonate are the of the is the be and the the hydrolysis of glutamine increases the of is to an an on glutamine it is that the of catalytic is in the of ATP and been C.J. Liao M. Biochemistry. 1993; 32: 1278-1284Crossref PubMed Scopus (19) Google Scholar, 30Miles B.W. Banzon J.A. Raushel F.M. Biochemistry. 1998; 37: 16773-16779Crossref PubMed Scopus (34) Google Scholar) for E. coli The functional linkage is in the Ser44 → Ala The of ATP and bicarbonate has no significant effect on or the of the that Ser44 is for of the that the GLN domain. activation an in the rate of of the it is that Ser44 but only ATP and bicarbonate are to the CPS domain. this functional linkage is in the reactions on the GLN and CPS then the rate of glutamine hydrolysis no longer the rate of carbamoyl phosphate synthesis the linkage is disrupted. The of carbamoyl phosphate synthesis is in the with 1 of glutamine of carbamoyl phosphate In contrast, the mutant hybrid hydrolyzed 3 times more glutamine than that needed for carbamoyl phosphate synthesis, with the presumably of the that in the GLN attenuation domain is not a catalytic residue in the but is an component in the regulatory linkage that phases glutamine hydrolysis and carbamoyl phosphate synthesis. Lusty for the of and
Hewagama et al. (Fri,) studied this question.