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• W1969333519 abstract "Two hybrid versions ofEscherichia coli aspartate transcarbamoylase were studied to determine the influence of domain closure on the homotropic and heterotropic properties of the enzyme. Each hybrid holoenzyme had one wild-type and one inactive catalytic subunit. In the first case the inactive catalytic subunit had Arg-54 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 17,000-fold reduction in activity, no loss in substrate affinity, and an R state structurally identical to that of the wild-type enzyme. In the second case, the inactive catalytic subunit had Arg-105 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 1,100-fold reduction in activity, substantial loss in substrate affinity, and loss of the ability to be converted to the R state. Thus, the R54A substitution results in a holoenzyme that can undergo closure of the catalytic chain domains to form the high activity, high affinity active site and to undergo the allosteric transition, whereas the R105A substitution results in a holoenzyme that can neither undergo domain closure nor the allosteric transition. The hybrid holoenzyme with one wild-type and one R54A catalytic subunit exhibited the same maximal velocity per active site as the wild-type holoenzyme, reduced cooperativity, and normal heterotropic interactions. The hybrid with one wild-type and one R105A catalytic subunit exhibited significantly reduced maximal velocity per active site as compared with the wild-type holoenzyme, reduced cooperativity, and substantially reduced heterotropic interactions. Small angle x-ray scattered was used to verify that the R105A-containing hybrid could attain an R state structure. These results indicate the global nature of the conformational changes associated with the allosteric transition in the enzyme. If one catalytic subunit cannot undergo domain closure to create the active sites, then the entire molecule cannot attain the high activity, high activity R state. Two hybrid versions ofEscherichia coli aspartate transcarbamoylase were studied to determine the influence of domain closure on the homotropic and heterotropic properties of the enzyme. Each hybrid holoenzyme had one wild-type and one inactive catalytic subunit. In the first case the inactive catalytic subunit had Arg-54 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 17,000-fold reduction in activity, no loss in substrate affinity, and an R state structurally identical to that of the wild-type enzyme. In the second case, the inactive catalytic subunit had Arg-105 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 1,100-fold reduction in activity, substantial loss in substrate affinity, and loss of the ability to be converted to the R state. Thus, the R54A substitution results in a holoenzyme that can undergo closure of the catalytic chain domains to form the high activity, high affinity active site and to undergo the allosteric transition, whereas the R105A substitution results in a holoenzyme that can neither undergo domain closure nor the allosteric transition. The hybrid holoenzyme with one wild-type and one R54A catalytic subunit exhibited the same maximal velocity per active site as the wild-type holoenzyme, reduced cooperativity, and normal heterotropic interactions. The hybrid with one wild-type and one R105A catalytic subunit exhibited significantly reduced maximal velocity per active site as compared with the wild-type holoenzyme, reduced cooperativity, and substantially reduced heterotropic interactions. Small angle x-ray scattered was used to verify that the R105A-containing hybrid could attain an R state structure. These results indicate the global nature of the conformational changes associated with the allosteric transition in the enzyme. If one catalytic subunit cannot undergo domain closure to create the active sites, then the entire molecule cannot attain the high activity, high activity R state. N-(phosphonoacetyl)-l-aspartate the aspartate concentration at half-maximal observed specific activity catalytic subunit of aspartate transcarbamoylase composed of three catalytic chains regulatory subunit of aspartate transcarbamoylase composed of two regulatory chains the mutant catalytic subunit in which 6 aspartate residues have been added to the C terminus of each catalytic chain the reconstituted mutant holoenzyme in which 6 aspartate residues have been added to the C terminus of each catalytic chain mutant hybrid holoenzyme in which one catalytic subunit has Ala substituted in place of Arg-105 in each catalytic chain and the other catalytic subunit has 6 aspartic acid residues added to the C terminus of each catalytic chain mutant hybrid holoenzyme in which one catalytic subunit has Ala substituted in place of Arg-54 in each catalytic chain and the other catalytic subunit has 6 aspartic acid residues added to the C terminus of each catalytic chain The homotropic cooperativity in Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) is directly related to the ability of the substrates to induce a structural and functional transition of the enzyme from a low activity low affinity T state to a high activity, high affinity R state (1Kantrowitz E.R. Lipscomb W.N. Trends Biochem. Sci. 1990; 15: 53-59Abstract Full Text PDF PubMed Scopus (122) Google Scholar). Structurally the conversion of the enzyme from the T to the R state involves an elongation of the enzyme by 11 Å along the molecular 3-fold axis along with a twist of the two catalytic subunits of about 5° in opposite directions (2Ke H.-M. Lipscomb W.N. Cho Y. Honzatko R.B. J. Mol. Biol. 1988; 204: 725-747Crossref PubMed Scopus (162) Google Scholar). This structural transition also results in a simultaneous rotation of each regulatory dimer about its own 2-fold axis. In addition to these quaternary structural changes, the T to R transition involves structural alterations on the tertiary level. For example, during the T to R transition each catalytic chain undergoes a domain closure in which the aspartate domain moves 3 Å toward the carbamoyl phosphate domain, resulting in a major reorganization of the interdomain interface. In addition to this tertiary rearrangement of domains, several loop regions in the catalytic chains undergo significant rearrangement. Specifically, the 80s loop in the carbamoyl phosphate domain rearranges from being held out of the active site in the T state to a new conformation that positions the side chains of Ser-80 and Lys-84 in contact with the substrates bound in the active site of an adjacent catalytic chain in the R state. The reorientation of the 240s loop containing residues 230–245 of the aspartate domain is intimately involved in the quaternary change. Residues within this loop region are involved in T state stabilizing interactions connecting the upper and lower catalytic subunits. These intersubunit interactions are absent in the R state, in which residues of this loop now form intrachain interactions with residues in the opposing carbamoyl phosphate domain (1Kantrowitz E.R. Lipscomb W.N. Trends Biochem. Sci. 1990; 15: 53-59Abstract Full Text PDF PubMed Scopus (122) Google Scholar). Probing of these interactions in earlier studies through site-specific mutagenesis has shown them to be critical in attaining the full R state conformation (3Ladjimi M.M. Kantrowitz E.R. Biochemistry. 1988; 27: 276-283Crossref PubMed Scopus (68) Google Scholar). In order to determine the influence of domain closure on the heterotropic and homotropic properties of aspartate transcarbamoylase in this work, we constructed two hybrid forms of the enzyme. Each of these hybrids has one wild-type catalytic subunit and one catalytic subunit in which each of the chains has a single amino acid substitution, rendering that subunit inactive. Therefore, by using these hybrid enzymes, we were able to measure the kinetic parameters due to the wild-type catalytic subunit within the context of the holoenzyme. In this fashion we can determine what effect if any the inactive subunit has on the wild-type subunit. The two inactive catalytic subunits used have mutations in the active site, R54A or R105A. As seen in Fig. 1, both Arg-54 and Arg-105 interact with the bisubstrate analog,N-phosphonoacetyl-l-aspartate (PALA),1 bound in the active site of the enzyme (4Jin L. Stec B. Lipscomb W.N. Kantrowitz E.R. Proteins Struct. Funct. Genet. 1999; 37: 729-742Crossref PubMed Scopus (73) Google Scholar). Although both the side chains of Arg-54 and Arg-105 interact with the carbamoyl phosphate portion of PALA, the kinetic consequences of these two mutations are quite different. Affinity for both carbamoyl phosphate and aspartate is little influenced by the R54A mutation, whereas the affinity for both substrates is reduced substantially for the R105A mutation (5Stebbins J.W., Xu, W. Kantrowitz E.R. Biochemistry. 1989; 28: 2592-2600Crossref PubMed Scopus (52) Google Scholar). The model of the proposed tetrahedral intermediate in the active site of the PALA-ligated structure (4Jin L. Stec B. Lipscomb W.N. Kantrowitz E.R. Proteins Struct. Funct. Genet. 1999; 37: 729-742Crossref PubMed Scopus (73) Google Scholar) reveals that the interactions for the Arg-105 side chain to the tetrahedral intermediate are similar to those observed to PALA but change dramatically for the Arg-54 side chain. For the Arg-54 side chain, interactions to the tetrahedral intermediate occur only with the anhydride oxygen, not present in PALA. Functionally, the R54A enzyme exhibits a 17,000-fold reduction in maximal velocity as opposed to the R105A enzyme that exhibits only a 1,100-fold reduction in maximal velocity (5Stebbins J.W., Xu, W. Kantrowitz E.R. Biochemistry. 1989; 28: 2592-2600Crossref PubMed Scopus (52) Google Scholar). The model of the tetrahedral intermediate (4Jin L. Stec B. Lipscomb W.N. Kantrowitz E.R. Proteins Struct. Funct. Genet. 1999; 37: 729-742Crossref PubMed Scopus (73) Google Scholar) suggests that the role of Arg-54 is to stabilize the negative charge on the phosphate leaving group and is not involved in substrate binding. Not only are these mutations different functionally but structurally as well. In both cases x-ray structures of the mutant holoenzymes have been determined in the presence of PALA. The structure of the R54A holoenzyme in the presence of PALA is virtually identical to that of the wild-type R state except at the site of the amino acid substitution (6Stebbins J.W. Robertson D.E. Roberts M.F. Stevens R.C. Lipscomb W.N. Kantrowitz E.R. Protein Sci. 1992; 1: 1435-1446Crossref PubMed Scopus (20) Google Scholar). The results for the R105A holoenzyme in the presence of PALA are much different. Although the enzyme was crystallized with 10 times as much PALA as the wild-type, the structure of the R105A enzyme was in the T state with no PALA bound at the active site (7Macol C.P. Tsuruta H. Stec B. Kantrowitz E.R. Nat. Struct. Biol. 2001; 8: 423-426Crossref PubMed Scopus (38) Google Scholar). Thus the amino acid substitution prevents the binding of PALA and the conversion of the enzyme into the R state. Hybrids constructed from one wild-type catalytic subunit and one R54A catalytic subunit, (R54A-C)(AT-C)R3, should interact with substrates in a much different fashion than the corresponding hybrid with the R105A catalytic subunit, (R105A-C)(AT-C)R3. In the case of the (R54A-C)(AT-C)R3 hybrid, the mutant catalytic subunit can bind substrates normally and undergo domain closure and the allosteric transition but contribute no significant catalytic activity, whereas in the case of the (R105A-C)(AT-C)R3 hybrid the mutant catalytic subunit cannot bind substrates and cannot undergo domain closure. These hybrids should be able to address directly the question of how independently the catalytic subunits function and whether the ability of one catalytic subunit to undergo domain closure can influence the function of the other catalytic subunit. Agarose, ATP, CTP, l-aspartate,N-carbamoyl-l-aspartate, potassium dihydrogen phosphate, and uracil were obtained from Sigma. Ampicillin, Tris, Q-Sepharose Fast Flow resin and Source Q resin were purchased fromAmersham Biosciences. Protein Assay Dye and SDS were purchased from Bio-Rad. Carbamoyl phosphate dilithium salt, obtained from Sigma, was purified before use by precipitation from 50% (v/v) ethanol and was stored desiccated at −20 °C (8Gerhart J.C. Pardee A.B. J. Biol. Chem. 1962; 237: 891-896Abstract Full Text PDF PubMed Google Scholar). Casamino acids, yeast extract, and tryptone were obtained from Difco. Ammonium sulfate and electrophoresis grade acrylamide were purchased from ICN Biomedicals (Costa Mesa, CA). Antipyrine was obtained from Fisher. The AT-C, R105A-C, and R54A-C catalytic subunits were overexpressed utilizing strain EK1104 (F− ara,thi, Δ(pro-lac), ΔpyrB,pyrF ±, rpsL) (9Nowlan S.F. Kantrowitz E.R. J. Biol. Chem. 1985; 260: 14712-14716Abstract Full Text PDF PubMed Google Scholar) containing plasmids pEK357 (10Sakash J.B. Chan R.S. Tsuruta H. Kantrowitz E.R. J. Biol. Chem. 2000; 275: 752-758Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), pEK99 (5Stebbins J.W., Xu, W. Kantrowitz E.R. Biochemistry. 1989; 28: 2592-2600Crossref PubMed Scopus (52) Google Scholar), and pEK78 (5Stebbins J.W., Xu, W. Kantrowitz E.R. Biochemistry. 1989; 28: 2592-2600Crossref PubMed Scopus (52) Google Scholar), respectively. Bacteria were cultured at 37 °C with agitation in M9 media (11Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1982: 368-369Google Scholar) containing 0.5% casamino acids, 12 μg/ml uracil, and 150 μg/ml ampicillin. Cells were harvested and resuspended in 0.1 m Tris-Cl buffer, pH 9.2, followed by sonication to lyse the cells. Two 65% ammonium sulfate fractionation steps were performed. Ion-exchange chromatography using Q-Sepharose Fast Flow resin was used to purify the enzyme further (12Stebbins J.W. Kantrowitz E.R. J. Biol. Chem. 1989; 264: 14860-14864Abstract Full Text PDF PubMed Google Scholar). After concentration, the purity of the enzymes was checked by SDS-PAGE (13Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205428) Google Scholar) and nondenaturing PAGE (14Davis B.J. Ann. N. Y. Acad. Sci. 1964; 121: 680-685Google Scholar, 15Ornstein L. Ann. N. Y. Acad. Sci. 1964; 121: 321-349Crossref PubMed Scopus (3318) Google Scholar). The aspartate transcarbamoylase regulatory subunit was overexpressed utilizing strain EK1104 containing plasmid pEK168 (10Sakash J.B. Chan R.S. Tsuruta H. Kantrowitz E.R. J. Biol. Chem. 2000; 275: 752-758Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Bacteria were cultured at 37 °C with agitation in M9 media (11Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1982: 368-369Google Scholar) containing 0.5% casamino acids, 12 μg/ml uracil, and 150 μg/ml ampicillin. Cells were harvested and resuspended in 0.1m Tris-Cl buffer, pH 9.2, 0.1 mm zinc chloride followed by sonication to lyse the cells. Two 65% ammonium sulfate fractionation steps were performed. As described previously, ion-exchange chromatography using Q-Sepharose Fast Flow resin was used to initially purify the regulatory subunit (16Dembowski N.J. Kantrowitz E.R. Protein Eng. 1993; 6: 123-127Crossref PubMed Scopus (7) Google Scholar). The regulatory chain was further purified by ion-exchange chromatography using Source-Q. The Source-Q (1 × 10 cm) column was equilibrated with 50 mm Tris acetate buffer, pH 8.3, 2 mm2-mercaptoethanol, and 0.1 mm zinc acetate at a flow rate of 1 ml/min. After the sample was loaded, the column was washed with 5 column volumes of 50 mm Tris acetate buffer, pH 8.3, 2 mm 2-mercaptoethanol, 0.1 mm zinc acetate. This was followed by a gradient (total volume of 10 column volumes) of 0–50% 50 mm Tris acetate buffer, pH 8.3, 2 mm2-mercaptoethanol, 0.1 mm zinc acetate, 1.0 mNaCl. After concentration of the appropriate fractions, the purity of the regulatory subunit was checked by SDS-PAGE (13Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205428) Google Scholar) and nondenaturing PAGE (14Davis B.J. Ann. N. Y. Acad. Sci. 1964; 121: 680-685Google Scholar, 15Ornstein L. Ann. N. Y. Acad. Sci. 1964; 121: 321-349Crossref PubMed Scopus (3318) Google Scholar). Equal amounts of purified R105A-C or R54A-C catalytic subunit and AT-C catalytic subunit were mixed with excess regulatory subunit and dialyzed overnight against 50 mm Tris acetate buffer, pH 8.3, 2 mm 2-mercaptoethanol, and 0.1 mm zinc acetate (17Sakash J.B. Kantrowitz E.R. Biochemistry. 1998; 37: 281-288Crossref PubMed Scopus (14) Google Scholar). The mixture was examined by nondenaturing PAGE to confirm the existence of the three holoenzyme species, (R105A-C)2R3, (R105A-C)(AT-C)R3, and (AT-C)2R3 or (R54A-C)2R3, (R54A-C)(AT-C)R3, and (AT-C)2R3. Separation of the species was performed using the UNO Q-1 column. The concentration of the wild-type regulatory subunit was determined from absorbance measurements at 280 nm using the extinction coefficients of 0.32 cm2 mg−1 (18Gerhart J.C. Holoubek H. J. Biol. Chem. 1967; 242: 2886-2892Abstract Full Text PDF PubMed Google Scholar). The concentrations of the mutant enzymes were determined by the Bio-Rad version of the Bradford dye-binding assay (19Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211664) Google Scholar). Nucleotide concentrations were determined at pH 7.0 from absorbance measurements at the λmax using the respective molar extinction coefficients of the nucleotides. The aspartate transcarbamoylase activity was measured at 25 °C by the colorimetric method (20Pastra-Landis S.C. Foote J. Kantrowitz E.R. Anal. Biochem. 1981; 118: 358-363Crossref PubMed Scopus (110) Google Scholar). Saturation curves were performed in duplicate, and data points shown in the figures are the average values. Assays were performed in 50 mm Tris acetate buffer, pH 8.3. Data analysis of the steady state kinetics was carried out as described previously (21Silver R.S. Daigneault J.P. Teague P.D. Kantrowitz E.R. J. Mol. Biol. 1983; 168: 729-745Crossref PubMed Scopus (45) Google Scholar). Fitting of the experimental data to theoretical equations was accomplished by non-linear regression. When substrate inhibition was negligible, data were fit to the Hill equation. If substrate inhibition was significant, data were analyzed using an extension of the Hill equation that included a term for substrate inhibition (22Pastra-Landis S.C. Evans D.R. Lipscomb W.N. J. Biol. Chem. 1978; 253: 4624-4630Abstract Full Text PDF PubMed Google Scholar). The nucleotide saturation curves were fit to a hyperbolic binding isotherm by non-linear regression. The small angle x-ray scattering experiments were performed on Beam Line 4–2 at the Stanford Synchrotron Radiation Laboratory (3.0 GeV, 50–100 mA). A significantly upgraded version of the small angle scattering instrument was used. The specimen-to-detector distance was 95 cm, and the x-ray wavelength was tuned to 1.381 Å using the Si(111) double-crystal monochromator (23Tsuruta H. Brennan S. Rek Z.U. Irving T.C. J. Appl. Crystallogr. 1998; 31: 672-682Crossref Scopus (54) Google Scholar). A linear gas chamber detector filled with a xenon/CO2mixture was used in the experiment. Total counting rate on the detector was between 30,000 and 90,000 counts/s. The scattering curves are expressed as the momentum transfer h (h = 4π(sinθ)/λ), where 2θ and λ are the scattering angle and the wavelength of the x-ray beam, respectively) which was calibrated using the (100) reflection from a cholesterol myristate powder sample held at the specimen position. Sample solutions were maintained at 25 °C. All scattering curves were normalized to incident beam intensity integrated over the exposure time, and the corresponding solvent scattering was subtracted. The enzyme solution was diluted so that all the scattering curves were performed at an identical protein concentration. The hybrid holoenzyme containing one AT-C catalytic subunit and one R54A-C catalytic subunit was generated by mixing R54A-C and AT-C catalytic subunits with excess regulatory subunit. The resulting species were identified by nondenaturing PAGE, and the (R54A-C)(AT-C)R3, (R54A-C)2R3, and (AT-C)2R3 holoenzymes were purified by anion-exchange chromatography (Fig. 2). Fig. 3 shows the aspartate saturation curves of the reconstituted hybrid holoenzymes, and the kinetic parameters calculated from these curves are given in TableI. The (R54A-C)(AT-C)R3holoenzyme exhibited a sigmoidal saturation curve with a Hill coefficient of 1.5. The lower Hill coefficient is expected due to the reduced number of active sites. The maximal velocity of the (R54A-C)(AT-C)R3 holoenzyme was 7.1 mmol·h−1·mg−1 and the [Asp]0.5 was 8.7 mm. This maximum velocity corresponds to 2.6 mmol·h−1·mg−1 per active site, the same value observed for the reconstituted (AT-C)2R3 enzyme.Figure 3Aspartate saturation curves of the (R105A-C)( AT-C )R3 (■), (R54A-C)( AT-C )R3(●), and ( AT-C )2R3 (○) holoenzymes.Velocity is reported in millimoles ofN-carbamoyl-l-aspartate formed per h per functional active site (see Table I). Colorimetric assays were performed at 25 °C at a saturating concentration of carbamoyl phosphate (4.8 mm) in 50 mm Tris acetate buffer, pH 8.3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IKinetic parameters for the (R105A-C)(AT-C)R3 and (R54A-C)(AT-C)R3 hybrid holoenzymesEnzymeFunctional active sitesMaximal velocity1-aValues are mmol · h−1 · mg−1.[Asp]0.5nHmmWild-type615.3 ± 0.78.4 ± 1.52.6 ± 0.3(R54A)2R3b60.001 261(R105A)2R3b60.0153101(R54A-C)(AT-C)R337.1 ± 0.88.7 ± 0.61.5 ± 0.2(R105A-C)(AT-C)R334.0 ± 0.57.9 ± 1.81.7 ± 0.2These data were determined from aspartate saturation curves. Colorimetric assays were performed at 25 °C in 0.05 mTris acetate buffer, pH 8.3, at a saturating concentration of carbamoyl phosphate (4.8 mm). The errors indicated are S.D. obtained from three or more determinations.1-bThe data previously determined (5Stebbins J.W., Xu, W. Kantrowitz E.R. Biochemistry. 1989; 28: 2592-2600Crossref PubMed Scopus (52) Google Scholar) for the mutant holoenzymes are presented for comparison.1-a Values are mmol · h−1 · mg−1. Open table in a new tab These data were determined from aspartate saturation curves. Colorimetric assays were performed at 25 °C in 0.05 mTris acetate buffer, pH 8.3, at a saturating concentration of carbamoyl phosphate (4.8 mm). The errors indicated are S.D. obtained from three or more determinations. 1-bThe data previously determined (5Stebbins J.W., Xu, W. Kantrowitz E.R. Biochemistry. 1989; 28: 2592-2600Crossref PubMed Scopus (52) Google Scholar) for the mutant holoenzymes are presented for comparison. The influence of the heterotropic effectors ATP and CTP on the (R54A-C)(AT-C)R3holoenzyme was determined at one-half the [Asp]0.5. This aspartate concentration was selected because the nucleotides exert a larger influence on the activity of the enzyme as the aspartate concentration is reduced (24Tauc P. Leconte C. Kerbiriou D. Thiry L. Hervé G. J. Mol. Biol. 1982; 155: 155-168Crossref PubMed Scopus (48) Google Scholar). Based upon these nucleotide saturation curves, the maximal extent of activation or inhibition at infinite nucleotide concentration was determined. As seen in Fig.4, ATP activates the (R54A-C)(AT-C)R3 holoenzyme 356%, essentially the same activation as observed for the wild-type enzyme. CTP inhibits the (R54A-C)(AT-C)R3 holoenzyme to a residual activity of 35%, slightly less than the 20% residual activity observed for the wild-type enzyme (Fig. 4). The hybrid (R105A-C)(AT-C)R3 was generated by mixing R105A-C and AT-C catalytic subunits with excess regulatory subunit. The resulting species were identified by nondenaturing PAGE, and the (R105A-C)(AT-C)R3 holoenzyme was purified by anion-exchange chromatography (data not shown). Fig. 3 shows the aspartate saturation curves of the reconstituted hybrid enzymes, and the kinetic parameters calculated from these curves are given in Table I. The (R105A-C)(AT-C)R3 holoenzyme exhibited a sigmoidal saturation curve with a Hill coefficient of 1.7, similar to the (R54A-C)(AT-C)R3 holoenzyme. The maximal velocity of (R105A-C)(AT-C)R3 holoenzyme was 4.0 mmol·h−1·mg−1 with an [Asp]0.5 of 7.9 mm. This maximum velocity corresponds to 1.3 mmol·h−1·mg−1 per active site, only one-half the activity observed for the (R54A-C)(AT-C)R3 and (AT-C)2R3holoenzymes. However, the [Asp]0.5 was approximately the same for all three species (see Fig. 3). The influence of the heterotropic effectors ATP and CTP on the (R105A-C)(AT-C)R3holoenzyme was determined at one-half the [Asp]0.5. Based upon these nucleotide saturation curves, the maximal extent of activation or inhibition at infinite nucleotide concentration was determined. As seen in Fig. 4, ATP activated the (R105A-C)(AT-C)R3 holoenzyme 164%, less than one-half the activation observed for the wild-type and (R54A-C)(AT-C)R3holoenzymes under the same conditions. CTP inhibited the (R105A-C)(AT-C)R3 holoenzyme to a residual activity of 85%, significantly less than the residual activity of 20% displayed by the wild-type enzyme and 35% residual activity displayed by the (R54A-C)(AT-C)R3 holoenzyme (Fig. 4). The influence of ATP was also determined at an aspartate concentration equal to four times [Asp]0.5. This concentration was used to determine whether the lower than expected activity observed for this holoenzyme was due to the inability of carbamoyl phosphate and aspartate to bring about the full T to R transition. If the substrates were unable to bring about the full T to R transition, ATP may still be able to activate the enzyme at saturating aspartate concentrations. However, no activation was observed at this higher aspartate concentration (data not shown). Small angle x-ray scattering has been used extensively in studying aspartate transcarbamoylase because this technique allows rapid determination of the quaternary state of the enzyme in solution (25Fetler L. Tauc P. Hervé G. Moody M.F. Vachette P. J. Mol. Biol. 1995; 251: 243-255Crossref PubMed Scopus (45) Google Scholar, 26Hervé G. Moody M.F. Tauc P. Vachette P. Jones P.T. J. Mol. Biol. 1985; 185: 189-199Crossref PubMed Scopus (83) Google Scholar). The scattering pattern of the wild-type enzyme in the absence of PALA is characteristic of the T-structural state, whereas the pattern in the presence of PALA is characteristic of the R-structural state. To determine whether the (R105A-C)(AT-C)R3holoenzyme could be converted to the R structural state, small angle x-ray scattering was performed in the absence and presence of PALA (Fig. 5). The change in scattering pattern observed upon addition of PALA to the hybrid enzyme established that it could be converted to the structural R state. The binding of PALA caused an increase in peak intensity and a shift in position of the first subsidiary maximum, two hallmarks of the T to R transition in wild-type aspartate transcarbamoylase (26Hervé G. Moody M.F. Tauc P. Vachette P. Jones P.T. J. Mol. Biol. 1985; 185: 189-199Crossref PubMed Scopus (83) Google Scholar). In the work reported here, two hybrid versions of aspartate transcarbamoylase were constructed and characterized to understand more fully how substrate binding and the subsequent tertiary movements in one catalytic subunit affect the other catalytic subunit in the holoenzyme, and how these tertiary movements are related to the global allosteric transition. To create the hybrid species, the wild-type catalytic subunit was engineered to carry six extra aspartic acid residues, which served as a chromatographic handle in the purification. Previous work (10Sakash J.B. Chan R.S. Tsuruta H. Kantrowitz E.R. J. Biol. Chem. 2000; 275: 752-758Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) has shown that the aspartate tail does not affect the structure or function of the catalytic subunit or the holoenzyme. The binding of aspartate to aspartate transcarbamoylase saturated with carbamoyl phosphate initiates a series of tertiary changes that induce the quaternary transition from the T to the R state (1Kantrowitz E.R. Lipscomb W.N. Trends Biochem. Sci. 1990; 15: 53-59Abstract Full Text PDF PubMed Scopus (122) Google Scholar). Within the catalytic chain the tertiary structural changes associated with the transition include the closure of the carbamoyl phosphate and aspartate domains along with major reorientations of the 80s and 240s loops (1Kantrowitz E.R. Lipscomb W.N. Trends Biochem. Sci. 1990; 15: 53-59Abstract Full Text PDF PubMed Scopus (122) Google Scholar). These conformational changes result in the repositioning of certain side chain residues that are critical for catalysis. In this study, we successfully applied the technology previously developed (10Sakash J.B. Chan R.S. Tsuruta H. Kantrowitz E.R. J. Biol. Chem. 2000; 275: 752-758Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) to create two hybrid enzymes, which allowed us to study how the aforementioned tertiary dynamics in one catalytic subunit affect catalysis and regulation in the other catalytic subunit of the holoenzyme. In each case, one of the two catalytic subunits is wild-type, whereas the other catalytic subunit has a single amino acid substitution that renders it inactive under the assay conditions used. The mutations, R54A and R105A, were selected because of the significantly different properties of the holoenzymes that have these mutations in all six catalytic chains (5Stebbins J.W., Xu, W. Kantrowitz E.R. Biochemistry. 1989; 28: 2592-2600Crossref PubMed Scopus (52) Google Scholar). Through comparing and contrasting of the kinetic properties of these two hybrid enzymes, we obtained a clearer view of how the two catalytic subunits of aspartate transcarbamoylase work in concert in the holoenzyme. Previous hybrid experiments have combined active and inactive subunits with wild-type regulatory subunits; however, these early hybrids were created and analyzed at a time when little structural data on the enzyme were available. As a result, the mutations made to inactivate one catalytic subunit most likely produced structural ramifications beyond the local site of the amino acid substitution or modification making the extension of the conclusions to the wild-type enzyme unclear. In their original hybrid study, Gibbons et al. (27Gibbons I. Yang Y.R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4452-4456Crossref PubMed Scopus (20) Google Scholar) created the CNCPR3 hybrid enzyme, which consisted of a native catalytic subunit, CN, a pyridoxylated catalytic subunit, CP, and native regulatory subunits, R. The pyridoxylation of the catalytic subunit was the result of a specific reaction involving reduction of a Schiff's base formed between pyridoxyl 5′-phosphate and a lysine side chain on each of the catalytic chains. Although the exact site of this pyridoxylation was not known at the time, kinetic characterization of the holoenzyme in which all six chains were modified in this manner indicated that the reaction effectively inactivated the enzyme. It is now known that the residue pyridoxylated is Lys-84, a residue located on the flexible 80s loop. The role of this loop as well as this specific residue has been extensively characterized (28Macol C. Dutta M. Stec B. Kantrowitz E.R. Protein Sci. 1999; 8: 1305-1313Crossref PubMed Scopus (7) Google Scholar). It has been shown that upon the T to R transition the 80s loop undergoes a dramatic rearrangement from being held out of the active site in the T state to being in the active site with residue 84 binding the substrate in the R state. If Lys-84 was modified by pyridoxylation, the R state conformation of active site would likely be unattainable with the 80s loop in an altered conformation in the CP subunit due to steric hindrance created by the pyridoxyl moiety. In another hybrid experiment, Gibbons et al. (29Gibbons I. Flatgaaard J.E. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 4298-4302Crossref PubMed Scopus (10) Google Scholar) created the CNCMR3 hybrid enzyme consisting of a native catalytic subunit, a catalytic subunit carrying a mutated residue, CM, and native regulatory subunits, R. In this case, the mutation was the result of a missense mutation. Again it was unknown at the time the exact location of the mutation; however, kinetic characterization verified that the alteration was sufficient to inactivate the CMCMR3 holoenzyme. Subsequent studies on the CMCMR3holoenzyme identified the mutation as a Gly-128 being changed to an aspartic acid (30Wall K.A. Schachman H.K. J. Biol. Chem. 1979; 254: 11917-11926Abstract Full Text PDF PubMed Google Scholar). Marked changes in the reactivity of several amino acid residues of the CMCMR3holoenzyme suggest that the single amino acid change in this location is sufficient to cause significant modification of the tertiary structure of the protein. Experimental data also revealed weakened subunit interactions in the mutant holoenzyme, suggesting an altered quaternary structure (31Wall K.A. Flatgaard J.E. Schachman H.K. Gibbons I. J. Biol. Chem. 1979; 254: 11910-11916Abstract Full Text PDF PubMed Google Scholar). The extraneous structural alterations in these hybrid systems prompted us to create carefully designed hybrids, which cause only the described local changes and thus could more accurately mimic the wild-type protein allowing an accurate assessment of how the two catalytic subunits of the molecule interact. As seen in Table I, both the (R54A-C)(AT-C)R3 and (R105A-C)(AT-C)R3 hybrid holoenzymes have [Asp]0.5 values nearly identical to that observed for the wild-type holoenzyme. In the case of the (R105A-C)(AT-C)R3enzyme, a higher [Asp]0.5 value would be expected should the R105A-C subunit contribute to the activity because of the extraordinarily high [Asp]0.5 induced by this mutation. Over the range of aspartate concentrations used in these experiments, the mutant catalytic subunit should contribute less than 0.33% of the total activity observed. Another similarity between the two hybrid holoenzymes is the observed decrease in cooperativity relative to the wild-type enzyme. This reduction is to be expected as the number of active sites has effectively been reduced by 50% and is reminiscent of the earlier active/inactive hybrids (27Gibbons I. Yang Y.R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4452-4456Crossref PubMed Scopus (20) Google Scholar). Striking differences between the two hybrid holoenzymes can be seen in the activity measured per active site and the regulation by the nucleotide effectors. The (R54A-C)(AT-C)R3 species behaves as would be expected for an enzyme with half the number of active sites. The activity per active site is essentially equal to the activity per active site in the wild-type enzyme. A lack of catalysis in one-half of the enzyme appears to have no deleterious effects on the functional active sites in the other catalytic subunit. Similarly, the nucleotide effectors, ATP and CTP, are able to activate and inhibit, respectively, the (R54A-C)(AT-C)R3 enzyme to nearly wild-type levels. In contrast, the lack of bound substrates in the R105A-C subunit has a significant and negative effect on the functional active sites within the (R105A-C)(AT-C)R3 hybrid. Without the substrates binding to the R105A-C active sites, the resulting lack of domain and loop movements prevents the full catalytic potential of the wild-type active sites in the context of this hybrid holoenzyme. Furthermore, the lack of substrate binding to half of the (R105A-C)(AT-C)R3 holoenzyme reduces the ability of the regulatory nucleotides to modulate catalytic activity. Could these results be due in part to a failure of the enzyme to undergo the T to R transition? Previously it has been shown that substrate binding at only a fraction of the active sites, in fact less than three of the active sites, is sufficient to allow the full T to R transition (7Macol C.P. Tsuruta H. Stec B. Kantrowitz E.R. Nat. Struct. Biol. 2001; 8: 423-426Crossref PubMed Scopus (38) Google Scholar). Small angle x-ray scattering experiments performed on the (R105A-C)(AT-C)R3 hybrid established that PALA can induce the full quaternary conformational change from the T to the R state, similar to that observed for the wild-type enzyme. Thus, a more likely explanation for the negative complementation effects observed for the (R105A-C)(AT-C)R3 hybrid is that the improper formation of the R state active sites in the mutant subunit has a global effect on the ability of the active sites in the wild-type catalytic subunit from attaining their correct R state conformation. The data presented in this study suggest that there are no distinct catalytic units within the holoenzyme. Based upon these data and previous functional (1Kantrowitz E.R. Lipscomb W.N. Trends Biochem. Sci. 1990; 15: 53-59Abstract Full Text PDF PubMed Scopus (122) Google Scholar) and structural studies (32Lipscomb W.N. Adv. Enzymol. 1994; 68: 67-151PubMed Google Scholar), we can propose a model for how the domain closure and loop motions in the catalytic chains are related to the allosteric transition. The binding of aspartate to the enzyme saturated with carbamoyl phosphate induces the closure of the two domains of that particular active site. This closure of the domains breaks specific interactions between the upper and lower catalytic subunits, such as the interaction between Glu-239 to both Tyr-164 and Lys-165, thereby destabilizing the T state of the enzyme. The closure of the domains of the catalytic chains is directly linked to the quaternary structural reorganization of the enzyme, because it is sterically impossible for the two domains of the catalytic chains to attain their full-closed conformation without the quaternary elongation of the molecule. The final conformation of each active site is then achieved by the reorganization of the loops necessary to create the active site (33Fetler L. Vachette P. Hervé G. Ladjimi M.M. Biochemistry. 1995; 34: 15654-15660Crossref PubMed Scopus (12) Google Scholar). Previous data have suggested that the domain closure caused by the binding of only one PALA molecule to the six active sites of the enzyme is sufficient to induce the global allosteric transition (7Macol C.P. Tsuruta H. Stec B. Kantrowitz E.R. Nat. Struct. Biol. 2001; 8: 423-426Crossref PubMed Scopus (38) Google Scholar). The data presented here also suggest that within the context of the holoenzyme, the domain closure and subsequent loop movements in each active site have a global and cumulative effect on the entire enzyme. The lack of substrate binding and domain closure in one subunit prevents the enzyme from being able to access the same structural R state as the wild-type enzyme. This study reinforces the importance of loop movement and domain closure for proper and efficient catalysis in aspartate transcarbamoylase. Stanford Synchrotron Radiation Laboratory is operated by the Department of Energy, Office of Basic Energy Sciences. The Stanford Synchrotron Radiation Laboratory Structural Biology Resource is supported by the National Institutes of Health, National Center for Research Resources Grant P41RR01209, and by the Department of Energy, Office of Biological and Environmental Research." @default.
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• W1969333519 title "Importance of Domain Closure for the Catalysis and Regulation ofEscherichia coli Aspartate Transcarbamoylase" @default.
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