Restricted Passage of Reaction Intermediates through the Ammonia Tunnel of Carbamoyl Phosphate Synthetase
2000; Elsevier BV; Volume: 275; Issue: 34 Linguagem: Inglês
10.1074/jbc.275.34.26233
ISSN1083-351X
AutoresXinyi Huang, Frank M. Raushel,
Tópico(s)Enzyme Structure and Function
ResumoThe x-ray crystal structure of the heterodimeric carbamoyl phosphate synthetase from Escherichia coli has identified an intermolecular tunnel that connects the glutamine binding site within the small amidotransferase subunit to the two phosphorylation sites within the large synthetase subunit. The tunneling of the ammonia intermediate through the interior of the protein has been proposed as a mechanism for the delivery of the ammonia from the small subunit to the large subunit. A series of mutants created within the ammonia tunnel were prepared by the placement of a constriction via site-directed mutagenesis. The degree of constriction within the ammonia tunnel of these enzymes was found to correlate to the extent of the uncoupling of the partial reactions, the diminution of carbamoyl phosphate formation, and the percentage of the internally derived ammonia that is channeled through the ammonia tunnel. NMR spectroscopy and a radiolabeled probe were used to detect and identify the enzymatic synthesis of N-amino carbamoyl phosphate and N-hydroxy carbamoyl phosphate from hydroxylamine and hydrazine. The kinetic results indicate that hydroxylamine, derived from the hydrolysis of γ-glutamyl hydroxamate, is channeled through the ammonia tunnel to the large subunit. Discrimination between the passage of ammonia and hydroxylamine was observed among some of these tunnel-impaired enzymes. The overall results provide biochemical evidence for the tunneling of ammonia within the native carbamoyl phosphate synthetase. The x-ray crystal structure of the heterodimeric carbamoyl phosphate synthetase from Escherichia coli has identified an intermolecular tunnel that connects the glutamine binding site within the small amidotransferase subunit to the two phosphorylation sites within the large synthetase subunit. The tunneling of the ammonia intermediate through the interior of the protein has been proposed as a mechanism for the delivery of the ammonia from the small subunit to the large subunit. A series of mutants created within the ammonia tunnel were prepared by the placement of a constriction via site-directed mutagenesis. The degree of constriction within the ammonia tunnel of these enzymes was found to correlate to the extent of the uncoupling of the partial reactions, the diminution of carbamoyl phosphate formation, and the percentage of the internally derived ammonia that is channeled through the ammonia tunnel. NMR spectroscopy and a radiolabeled probe were used to detect and identify the enzymatic synthesis of N-amino carbamoyl phosphate and N-hydroxy carbamoyl phosphate from hydroxylamine and hydrazine. The kinetic results indicate that hydroxylamine, derived from the hydrolysis of γ-glutamyl hydroxamate, is channeled through the ammonia tunnel to the large subunit. Discrimination between the passage of ammonia and hydroxylamine was observed among some of these tunnel-impaired enzymes. The overall results provide biochemical evidence for the tunneling of ammonia within the native carbamoyl phosphate synthetase. carbamoyl phosphate synthetase 2-(cyclohexylamino)ethanesulfonic acid Carbamoyl phosphate synthetase (CPS)1 from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP (Equation 1) (1Anderson P.M. Meister A. Biochemistry. 1965; 4: 2803-2809Crossref PubMed Scopus (111) Google Scholar). When one or more of these substrates are not present, CPS catalyzes three partial reactions: (a) the hydrolysis of glutamine, (b) the bicarbonate-dependent hydrolysis of ATP, and (c) the formation of ATP from carbamoyl phosphate and ADP (Equations Equation 2, Equation 3, Equation 4) (2Anderson P.M. Meister A. Biochemistry. 1966; 5: 3164-3169Crossref PubMed Scopus (85) Google Scholar). Ammonia can substitute for glutamine in the overall reaction as an alternative source of nitrogen (Equation 5). The enzyme is composed of a small amidotransferase subunit and a large synthetase subunit (1Anderson P.M. Meister A. Biochemistry. 1965; 4: 2803-2809Crossref PubMed Scopus (111) Google Scholar, 3Matthews S.L. Anderson P.M. Biochemistry. 1972; 11: 1176-1183Crossref PubMed Scopus (51) Google Scholar, 4Trotta 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). The synthesis of carbamoyl phosphate has been postulated to occur via four distinct chemical steps within the active sites of CPS (Scheme FS1) (2Anderson P.M. Meister A. Biochemistry. 1966; 5: 3164-3169Crossref PubMed Scopus (85) Google Scholar). The x-ray structure of CPS from E. coli has confirmed that the binding site for glutamine is contained within the small subunit, whereas the catalytic sites for the phosphorylation of bicarbonate and carbamate are located within the large subunit (5Thoden J.B. Holden H.M. Wesenberg G. Raushel F.M. Rayment I. Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (302) Google Scholar, 6Thoden J.B. Raushel F.M. Benning M.M. Rayment I. Holden H.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 8-24Crossref PubMed Scopus (85) Google Scholar). However, the most intriguing feature of the CPS structure is that the three active sites located within the small and large subunits are physically separated in three-dimensional space by nearly 100 Å (5Thoden J.B. Holden H.M. Wesenberg G. Raushel F.M. Rayment I. Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (302) Google Scholar, 6Thoden J.B. Raushel F.M. Benning M.M. Rayment I. Holden H.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 8-24Crossref PubMed Scopus (85) Google Scholar). The channeling of ammonia and carbamate through the interior of the protein has been proposed as a mechanism to deliver these two labile intermediates from one active site to the next (7Raushel F.M. Thoden J.B. Reinhart G.D. Holden H.M. Cur. Opin. Chem. Biol. 1998; 2: 624-632Crossref PubMed Scopus (25) Google Scholar, 8Holden H.M. Thoden J.B. Raushel F.M. Cur. Opin. Struct. Biol. 1998; 8: 679-685Crossref PubMed Scopus (39) Google Scholar, 9Raushel F.M. Thoden J.B. Holden H.M. Biochemistry. 1999; 38: 7891-7899Crossref PubMed Scopus (91) Google Scholar). For recent reviews on substrate channeling, see Refs. 10Ovadi J. J. Theor. Biol. 1991; 152: 1-22Crossref PubMed Scopus (193) Google Scholar, 11Miles E.W. Rhee S. Davies D.R. J. Biol. Chem. 1999; 274: 12193-12196Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 12Anderson K.S. Methods Enzymol. 1999; 308: 111-145Crossref PubMed Scopus (65) Google Scholar. 2MgATP+HCO3−+Gln+H2O→2MgADP+Pi+Glu+carbamoylPEquation 1 Gln+H2O→Glu+NH3Equation 2 MgATP+H2O→MgADP+PiEquation 3 MgADP+carbamoylP→MgATP+NH2CO2−Equation 4 2MgATP+HCO3−+NH3→2MgADP+Pi+carbamoylPEquation 5 Utilizing a visual inspection of the CPS model and a computational search with the software package GRASP, Thoden et al. (5Thoden J.B. Holden H.M. Wesenberg G. Raushel F.M. Rayment I. Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (302) Google Scholar, 6Thoden J.B. Raushel F.M. Benning M.M. Rayment I. Holden H.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 8-24Crossref PubMed Scopus (85) Google Scholar) identified a molecular tunnel within the interior of the heterodimeric protein, which leads from the glutamine binding site within the small subunit toward the two phosphorylation sites within the large subunit. Consequently, the ammonia produced within the active site of the small subunit must transverse the first half of this molecular tunnel in order to react with the carboxy phosphate intermediate formed at the site of bicarbonate phosphorylation within the large subunit. The carbamate intermediate must then diffuse through the second half of the molecular tunnel to be phosphorylated by the ATP bound to the other phosphorylation site of the large subunit. A ribbon representation of CPS with the relative locations of the three active sites and the intermolecular tunnel is shown in Fig. 1(5Thoden J.B. Holden H.M. Wesenberg G. Raushel F.M. Rayment I. Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (302) Google Scholar).Figure 1An α-carbon trace of the three-dimensional structure of CPS from E. coli(taken from Ref. 5Thoden J.B. Holden H.M. Wesenberg G. Raushel F.M. Rayment I. Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (302) Google Scholar). The binding sites for glutamine, ATP/bicarbonate, and carbamoyl phosphate are highlighted. The molecular tunnel connecting the three binding sites isoutlined in red.View Large Image Figure ViewerDownload (PPT)Biochemical results are fully consistent with the tunneling of the ammonia and carbamate intermediates. Isotope competition experiments with 15NH3 and unlabeled glutamine have demonstrated that the internal ammonia, derived directly from the hydrolysis of glutamine, does not dissociate from the small subunit and then reassociate to the large subunit (13Mullins L.S. Raushel F.M. J. Am. Chem. Soc. 1999; 121: 3803-3804Crossref Scopus (32) Google Scholar). The pH activity profiles also confirm that the enzyme-bound NH3 must be sequestered from the bulk solvent because NH4+ is not a substrate for the synthetase reaction (14Rubino S.D. Nyunoya H. Lusty C.J. J. Biol. Chem. 1986; 261: 11320-11327Abstract Full Text PDF PubMed Google Scholar, 15Cohen N.S. Kyan F.S. Jyan S.S. Cheung C.W. Raijman L. Biochem. J. 1985; 229: 205-211Crossref PubMed Scopus (58) Google Scholar). These results are thus consistent with a mechanism that requires the direct tunneling of ammonia between the two subunits. The lack of an 18O isotope exchange reaction between solvent water and bicarbonate during the overall synthesis of carbamoyl phosphate suggests that all of the carbon-containing intermediates (carboxy phosphate and carbamate) are fully committed to the formation of carbamoyl phosphate and not subjected to hydrolysis (16Raushel F.M. Mullins L.S. Gibson G.E. Biochemistry. 1998; 37: 10272-10278Crossref PubMed Scopus (17) Google Scholar). Moreover, the half-life of 70 ms for carbamate at neutral pH (17Wang T.T. Bishop S.H. Himoe A. J. Biol. Chem. 1972; 247: 4437-4440Abstract Full Text PDF PubMed Google Scholar) renders it highly improbable that carbamate would be able to dissociate from the first phosphorylation site and then reassociate to the second phosphorylation site within the large subunit. These results thus support the tunneling of carbamate between the two active sites contained within the large subunit.We have previously altered some of the residues that define the interior walls of the ammonia tunnel within the small subunit with the intention of providing more direct experimental support for the functional significance of the molecular tunnel within CPS (18Huang X. Raushel F.M. Biochemistry. 2000; 39: 3240-3247Crossref PubMed Scopus (38) Google Scholar). Two mutant proteins (G359Y and G359F) displayed kinetic properties consistent with a constriction or blockage of the ammonia tunnel. With the wild-type CPS, the hydrolysis of ATP and glutamine is fully coupled to one another such that one equivalent of carbamoyl phosphate is formed when two equivalents of ATP and one equivalent of glutamine are hydrolyzed. However, with both of these mutants, the hydrolysis of ATP and glutamine became uncoupled from the synthesis of carbamoyl phosphate. However, these mutants were fully functional when external ammonia was utilized as the nitrogen source, even though these proteins were unable to use glutamine for the synthesis of carbamoyl phosphate. These results also suggested the existence of an alternate route to the bicarbonate phosphorylation site when ammonia is provided as an external nitrogen source (18Huang X. Raushel F.M. Biochemistry. 2000; 39: 3240-3247Crossref PubMed Scopus (38) Google Scholar).In order to more fully substantiate the direct tunneling of the ammonia intermediate as a mechanistic link within the CPS heterodimer, we have now made further refinements to the architecture of the ammonia tunnel. In the first approach, we modulated the size of the physical constriction within the ammonia tunnel located in the small subunit of carbamoyl phosphate synthetase. This was attempted through the placement of hydrophobic side chains of varying sizes within the walls of the ammonia tunnel. In the second approach, hydroxylamine and hydrazine were utilized as alternative nitrogen sources of greater bulk. If hydroxylamine and hydrazine are able to be transported through the ammonia tunnel of the wild-type CPS, then the variable physical constrictions within the ammonia tunnel may differentially affect the transport rate from one active site to the next.RESULTSThe previous investigation of the ammonia tunnel of CPS demonstrated that the passage of ammonia could be restricted within the tunnel via the substitution of specific residues, which constitute part of the tunnel wall, with bulkier hydrophobic residues (18Huang X. Raushel F.M. Biochemistry. 2000; 39: 3240-3247Crossref PubMed Scopus (38) Google Scholar). In order to more fully substantiate the tunneling of the ammonia intermediate within CPS, a new series of mutants with different degrees of potential constriction within the ammonia tunnel was created. Three mutants, G359S, G359L, and S35F, were constructed and purified in addition to the wild-type CPS and the previously prepared G359F. The wild-type and mutant enzymes were expressed and purified to greater than 95% homogeneity, as judged by SDS-polyacrylamide gel electrophoresis. The effects of these modifications on the catalytic properties of CPS were determined for each mutant by measuring the rate of carbamoyl phosphate synthesis and the rates of the partial reactions. The kinetic parameters, K m andk cat, obtained for the wild-type and the mutant enzymes, are summarized in TablesTable I, Table II, Table III.Table IKinetic parameters for the ATPase reactions of the wild-type and mutant enzymesEnzymesHCO3−- dependent1-aReaction conditions for the bicarbonate-dependent ATPase reaction: pH 7.6, 25 °C, 5.0 mm ATP, 40 mm bicarbonate, 20 mm MgCl2, 100 mm KCl, and 10 mm ornithine.NH3-dependent1-bReaction conditions: the same as above except for variable amounts of NH4Cl.Gln-dependent1-cReaction conditions: the same as above except for variable amounts of glutamine.k catk catK m (NH4+)k catK m (Gln)s −1s −1mms −1mmWild-type0.39 ± 0.017.0 ± 0.6250 ± 325.8 ± 0.10.098 ± 0.004S35F0.49 ± 0.025.4 ± 0.2231 ± 255.7 ± 0.10.46 ± 0.01G359S1.2 ± 0.112 ± 0.234 ± 23.2 ± 0.320 ± 6G359L1.2 ± 0.110.5 ± 0.331 ± 31.51-dRate constant at a glutamine concentration of 40 mm.NA1-eNA, not applicable.G359F1-fData from Ref. 15.1.2 ± 0.112 ± 0.234 ± 11.51-dRate constant at a glutamine concentration of 40 mm.NAShown are kinetic constants for ADP formation monitored for the bicarbonate-dependent ATPase (Equation 3), ammonia-dependent ATPase (Equation 5), or glutamine-dependent ATPase reactions (Equation 1).1-a Reaction conditions for the bicarbonate-dependent ATPase reaction: pH 7.6, 25 °C, 5.0 mm ATP, 40 mm bicarbonate, 20 mm MgCl2, 100 mm KCl, and 10 mm ornithine.1-b Reaction conditions: the same as above except for variable amounts of NH4Cl.1-c Reaction conditions: the same as above except for variable amounts of glutamine.1-d Rate constant at a glutamine concentration of 40 mm.1-e NA, not applicable.1-f Data from Ref. 15Cohen N.S. Kyan F.S. Jyan S.S. Cheung C.W. Raijman L. Biochem. J. 1985; 229: 205-211Crossref PubMed Scopus (58) Google Scholar. Open table in a new tab Table IIKinetic parameters for the glutaminase reaction of the wild-type and mutant enzymesEnzymesPartial reaction2-aReaction conditions: pH 7.6, 25 °C, variable amounts of glutamine, 100 mm KCl.Overall reaction2-bReaction conditions: pH 7.6, 25 °C, variable amounts of glutamine, 5.0 mm ATP, 40 mm bicarbonate, 20 mm MgCl2, 100 mm KCl, and 10 mm ornithine.k catK m (Gln)k catK m (Gln)s −1mms −1mmWild-type0.0040 ± 0.00010.083 ± 0.0062.9 ± 0.10.069 ± 0.007S35F0.0015 ± 0.00010.084 ± 0.0135.6 ± 0.10.44 ± 0.03G359S0.033 ± 0.00122 ± 25.0 ± 0.229 ± 2G359L0.028 ± 0.00124 ± 22.5 ± 0.124 ± 2G359F2-cData from Ref. 15.0.0282-dRate constant at 40 mmglutamine.>402.42-dRate constant at 40 mmglutamine.>40Shown are kinetic constants for glutamate formation in the absence (Equation 2) or presence (Equation 1) of ATP and bicarbonate.2-a Reaction conditions: pH 7.6, 25 °C, variable amounts of glutamine, 100 mm KCl.2-b Reaction conditions: pH 7.6, 25 °C, variable amounts of glutamine, 5.0 mm ATP, 40 mm bicarbonate, 20 mm MgCl2, 100 mm KCl, and 10 mm ornithine.2-c Data from Ref. 15Cohen N.S. Kyan F.S. Jyan S.S. Cheung C.W. Raijman L. Biochem. J. 1985; 229: 205-211Crossref PubMed Scopus (58) Google Scholar.2-d Rate constant at 40 mmglutamine. Open table in a new tab Table IIIKinetic parameters for carbamoyl phosphate synthesis by the wild-type and mutant enzymesEnzymesNH3-dependent3-aReaction conditions: pH 7.6, 25 °C, variable amounts of NH4Cl, 5 mm ATP, 40 mm bicarbonate, 20 mm, MgCl2, 100 mm, KCl, 10 mm ornithine.Gln-dependent3-bReaction conditions: pH 7.6, 25 °C, variable amounts of glutamine, 5.0 mm ATP, 40 mm bicarbonate, 20 mm MgCl2, 100 mm KCl, and 10 mm ornithine.k catK m (NH4Cl)k catK m (Gln)s −1mms −1mmWild-type2.9 ± 0.1211 ± 233.2 ± 0.10.075 ± 0.003S35F3.2 ± 0.2265 ± 292.4 ± 0.10.43 ± 0.02G359S6.3 ± 0.228 ± 21.1 ± 0.127 ± 6G359L5.6 ± 0.125 ± 20.14 ± 0.0230 ± 10G359F3-cData from Ref. 15.4.8 ± 0.227 ± 30.0493-dRate constant at 40 mmglutamine.>40Shown are kinetic constants for carbamoyl phosphate formation (Equations 5 and 1).3-a Reaction conditions: pH 7.6, 25 °C, variable amounts of NH4Cl, 5 mm ATP, 40 mm bicarbonate, 20 mm, MgCl2, 100 mm, KCl, 10 mm ornithine.3-b Reaction conditions: pH 7.6, 25 °C, variable amounts of glutamine, 5.0 mm ATP, 40 mm bicarbonate, 20 mm MgCl2, 100 mm KCl, and 10 mm ornithine.3-c Data from Ref. 15Cohen N.S. Kyan F.S. Jyan S.S. Cheung C.W. Raijman L. Biochem. J. 1985; 229: 205-211Crossref PubMed Scopus (58) Google Scholar.3-d Rate constant at 40 mmglutamine. Open table in a new tab Kinetic Properties of S35FThe substitution of Ser-35 with a bulky phenylalanine was intended to block or constrict the passage of ammonia to the large subunit within the ammonia tunnel. The catalytic properties of the S35F mutant are very similar to the wild-type enzyme, with two notable exceptions. During the overall synthesis of carbamoyl phosphate with glutamine as the nitrogen source, theK m values for glutamine are elevated by 5–6-fold (Tables Table I, Table II, Table III), and the maximal rate of glutamine hydrolysis is about twice the wild-type value (Table II). Consequently, the partial reactions within the heterodimer of S35F are mildly uncoupled from one another. An estimate of reaction stoichiometry is obtained from the ratio of the maximal rate of the formation of ADP (Table I), glutamine (Table II), and carbamoyl phosphate (Table III). For the synthesis of one equivalent of carbamoyl phosphate, 2.4 equivalents of ATP and 2.3 equivalents of glutamine are consumed.Kinetic Properties of G359SThe replacement of Gly-359 with a serine residue yields a mutant of CPS with properties somewhat between the wild-type protein and G359F. With G359F, the hydrolysis of ATP and glutamine became almost completely uncoupled from the synthesis of carbamoyl phosphate (18Huang X. Raushel F.M. Biochemistry. 2000; 39: 3240-3247Crossref PubMed Scopus (38) Google Scholar). With G359S, it is apparent that these two partial reactions are still coupled to one another to a significant extent, as evidenced by the formation of carbamoyl phosphate and the mutual stimulation of the bicarbonate-dependent ATPase and glutaminase reactions. In all of the glutamine-dependent reactions, the K m values for glutamine are elevated by 200–400-fold (Tables Table I, Table II, Table III). During the synthesis of carbamoyl phosphate, the rate of glutamine hydrolysis is enhanced by 150-fold (Table II) relative to the absence of ATP and bicarbonate. The mutant also has an elevated bicarbonate-dependent ATPase activity, which is further stimulated about 3-fold in the presence of glutamine (Table I). When glutamine is used as the nitrogen source, the maximal rate of carbamoyl phosphate formation is one-third of the wild-type value (Table III), and 3 equivalents of ATP and 5 equivalents of glutamine are utilized for the production of one equivalent of carbamoyl phosphate.Kinetic Properties of G359LGly-359 was also mutated to a leucine residue. Although G359L retains the ability to enhance the basal glutaminase activity in the presence of ATP and bicarbonate (88-fold), it no longer stimulates the bicarbonate-dependent hydrolysis of ATP in the presence of glutamine (Tables I and II). This uncoupling between the partial reactions is also reflected in the inability to produce significant amounts of carbamoyl phosphate. The rate of carbamoyl phosphate formation with G359L is less than 5% that of the wild-type CPS (TableIII), and 11 equivalents of ATP and 18 equivalents of glutamine are required for the synthesis of one equivalent of carbamoyl phosphate. The overall reaction stoichiometry of this series of tunnel mutants is summarized in Fig. 2. The mutants at position-359 are fully functional when external ammonia is provided as the alternate nitrogen source. It is unclear why thek cat values are slightly higher than the wild-type value. Moreover, the K m values for NH4+/NH3 are reduced by 8–11-fold, whereas the wild-type reaction stoichiometry is preserved (Table III).Figure 2The reaction stoichiometry of the wild-type and mutant enzymes. The products, ADP, glutamate, and carbamoyl phosphate, are represented by the black, white, andgray bars, respectively. The amount of carbamoyl phosphate is normalized as 1 for each enzyme. The reaction stoichiometry is obtained from the ratio of the maximal rate of the formation of ADP (Table I), glutamine (Table II), and carbamoyl phosphate (Table III). The reaction stoichiometry of the wild-type (WT) CPS is 2 ATP:1 glutamine:1 carbamoyl phosphate.View Large Image Figure ViewerDownload (PPT)Enzymatic Formation of N-Hydroxy Carbamoyl Phosphate and N-Amino Carbamoyl PhosphateAn alternative approach to the placement of a constriction within the ammonia tunnel of CPS is the utilization of nucleophiles other than ammonia as the nitrogen source. Both hydroxylamine and hydrazine are bulkier than ammonia. Attempts were made to detect the formation of carbamoyl phosphate analogs from these two alternative nitrogen sources. Hydroxylamine, hydrazine, or ammonia was incubated with MgATP, [14C]bicarbonate, and CPS. The reactions were quenched at various times, and the unreacted bicarbonate was removed. The amount of nonvolatile 14C-containing product(s) was estimated by liquid scintillation counting (TableIV). For the reaction with ammonia, this method provides a quantitative assay for carbamoyl phosphate (27Miles B.W. Raushel F.M. Biochemistry. 2000; 39: 5051-5056Crossref PubMed Scopus (32) Google Scholar). The results here are thus consistent with the formation of a carbamoyl phosphate-like product in the enzymatic reaction using hydroxylamine or hydrazine as the nitrogen source. Similar results were also obtained when γ-glutamyl hydroxamate or γ-glutamyl hydrazide was provided as the nitrogen source (Table IV). These carbamoyl phosphate-like products have been identified as N-hydroxy carbamoyl phosphate (Scheme FS2, panel 2) andN-amino carbamoyl phosphate (Scheme FS2, panel 3) by NMR spectroscopy using hydroxylamine and hydrazine as nitrogen sources. The 13C and 31P NMR spectra of these two carbamoyl phosphate analogs are very similar to those of carbamoyl phosphate (Fig. 3). For all three compounds, the coupling constants between the carbonyl carbon and the phosphorus atom are close to 5 Hz, and the coupling constants between the carbonyl carbon and the amide nitrogen are about 25–26 Hz (Fig.3). In every case, the phosphorus atom did not appear coupled to the amide nitrogen. For N-amino carbamoyl phosphate (3Matthews S.L. Anderson P.M. Biochemistry. 1972; 11: 1176-1183Crossref PubMed Scopus (51) Google Scholar), no coupling between the carbonyl carbon and the terminal amino group was observed. It has been reported that the coupling constant between the methyl carbon and the terminal amino nitrogen inN,N-[13C]dimethyl[15N]hydrazine is less than 1 Hz (28Lichter R.L. Roberts J.D. J. Am. Chem. Soc. 1970; 93: 5218-5224Crossref Scopus (121) Google Scholar). Attempts to obtain the 15N NMR spectra of the two carbamoyl phosphate analogs were unsuccessful because of the negative nuclear Overhauser effects, proton exchange, and pH fluctuations during the data acquisition. The 13C and 31P signals of the two carbamoyl phosphate analogs were not due to contamination by ammonia in the hydroxylamine or hydrazine. Although carbamoyl phosphate was fully converted to citrulline by ornithine transcarbamoylase according to the 13C and31P NMR spectra, N-hydroxyl carbamoyl phosphate and N-amino carbamoyl phosphate were not substrates for ornithine transcarbamoylase. Second, although carbamoyl phosphate was more stable at neutral pH than alkaline pH, both analogs of carbamoyl phosphate appeared more stable at alkaline pH.Table IVEnzymatic formation of nonvolatile 14 C-containing products from various nitrogen sources, [ 14 C]bicarbonate, and ATPNitrogen sourceProduct5 min incubation10 min incubationμmControl0.40.5NH4Cl11941851NH2OH49.876.8NH2NH223.351.7Glutamine24102550γ-glutamyl hydroxamate41.648.9γ-Glutamyl hydrazide15.723.9The formation of carbamoyl phosphate or analogs from various nitrogen sources was probed using [14C]bicarbonate. Each reaction mixture contained 30 mm ATP, 5 mm[14C]bicarbonate (specific activity of 24 μCi/μmol) and 16 μm CPS. The nitrogen sources were 300 mmNH4Cl, 300 mm NH2OH, 300 mmNH2NH2, 20 mm glutamine, 20 mmγ-glutamyl hydroxamate, or 20 mm γ-glutamyl hydrazide. A control reaction containing 300 mm NH4Cl, which contained no CPS, was carried out in parallel. Aliquots of the reaction mixtures were acid quenched after 5 and 10 min. The unreacted bicarbonate was removed by the addition of powered dry ice. The amount of the nonvolatile product was determined by liquid scintillation counting. Open table in a new tab Figure FS2View Large Image Figure ViewerDownload (PPT)Figure 3NMR spectra of carbamoyl phosphate and analogs. The 13C and 31P NMR spectra for the control (1), carbamoyl phosphate (2),N-hydroxy carbamoyl phosphate (3), andN-amino carbamoyl phosphate (4) are shown.A and B present the 13C NMR spectra, and C presents the 31P NMR spectra. ForA and C, [13C]bicarbonate and [14N]nitrogen sources were used. For B,[13C]bicarbonate and 15N-enriched nitrogen sources were used. The 31P NMR spectra of the doubly enriched products were identical to those shown in panel C. Carbamoyl phosphate: δ (13C NMR, D2O) 156.85, δ (31P NMR, D2O) –4.35, JC,P = 4.7 Hz, JC,NH2CO = 25.7 Hz; N-hydroxy carbamoyl phosphate: δ (13C NMR, D2O) 156.90, δ (31P NMR, D2O) –4.50, JC,P = 5.0 Hz, JC, NHCO = 25.2 Hz; N-amino carbamoyl phosphate: δ (13C NMR, D2O) 156.72, δ (31P NMR, D2O) –4.12, JC,P = 4.7 Hz, JC, NHCO = 26.3 Hz.View Large Image Figure ViewerDownload (PPT)γ-Glutamyl Hydroxamate and γ-Glutamyl Hydrazide as Substrates of Wild-type CPSThe hydrolysis of γ-glutamyl hydroxamate and γ-glutamyl hydrazide by CPS has been reported (14Rubino S.D. Nyunoya H. Lusty C.J. J. Biol. Chem. 1986; 261: 11320-11327Abstract Full Text PDF PubMed Google Scholar, 19Anderson P.M. Meister A. Biochemistry. 1966; 5: 3157-3163Crossref PubMed Scopus (93) Google Scholar). In the absence of ATP and bicarbonate, the kinetic parameters for the hydrolysis of γ-glutamyl hydroxamate and γ-glutamyl hydrazide are very similar to that for glutamine except that theK m value for the hydrazide is 5-fold higher than theK m for glutamine (TableV). In the presence of saturating levels of ATP and bicarbonate, the maximal rate for the hydrolysis of γ-glutamyl hydroxamate is enhanced 2700-fold, whereas theK m value for γ-glutamyl hydroxamate is increased 13-fold relative to the absence of ATP and bicarbonate (Table V). For comparison, the enhancement in the k cat value with glutamine is about 700-fold, whereas the K m value for glutamine is unchanged (Table II). For the hydrolysis of γ-glutamyl hydrazide in the presence of ATP and bicarbonate, thek cat value is enhanced by more than 100-fold relative to the absence of ATP and bicarbonate. However, there is little net enhancement ink cat /K m because theK m value for γ-glutamyl hydrazide is also increased by the same extent.Table VKinetic parameters for the hydrolysis of γ-glutamyl hydroxamate and γ-glutamyl hydrazide by the wild-type and mutant enzymesγ-Glutamyl hydroxamateγ-Glutamyl hydrazidePartial reaction5-aReaction conditions: pH 7.6, 25 °C, 100 mm KCl, variable amounts of γ-glutamyl hydroxamate, or γ-glutamyl hydrazide.Overall reaction5-bReaction conditions: pH 7.6, 25 °C, variable amounts of γ-glutamyl hydroxamate or γ-glutamyl hydrazide, 5.0 mm ATP, 40 mm bicarbonate, 20 mm MgCl2, 100 mm KCl, and 10 mm ornithine.Partial reaction5-aReaction conditions: pH 7.6, 25 °C, 100 mm KCl, variable amounts of γ-glutamyl hydroxamate, or γ-glutamyl hydrazide.Overall reaction5-bReaction conditions: pH 7.6, 25 °C, variable amounts of γ-glutamyl hydroxamate or
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