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The Contribution of Individual Interchain Interactions to the Stabilization of the T and R States of Escherichia coliAspartate Transcarbamoylase

2000; Elsevier BV; Volume: 275; Issue: 37 Linguagem: Inglês

10.1074/jbc.m005079200

1083-351X

Jessica B. Sakash, Evan R. Kantrowitz,

Metabolism and Genetic Disorders

Stabilization of the T and R allosteric states ofEscherichia coli aspartate transcarbamoylase is governed by specific intra- and interchain interactions. The six interchain interactions between Glu-239 in one catalytic chain of one catalytic trimer with both Lys-164 and Tyr-165 of a different catalytic chain in the other catalytic trimer have been shown to be involved in the stabilization of the T state. In this study a series of hybrid versions of aspartate transcarbamoylase was studied to determine the minimum number of these Glu-239 interactions necessary to maintain homotropic cooperativity and the T allosteric state. Hybrids with zero, one, and two Glu-239 stabilizing interactions do not exhibit cooperativity, whereas the hybrids with three or more Glu-239 stabilizing interactions exhibit cooperativity. The hybrid enzymes with one or more of the Glu-239 stabilizing interactions also exhibit heterotropic interactions. Two hybrids with three Glu-239 stabilizing interactions, in different geometric relationships, had identical properties. From this and previous studies, it is concluded that the 239 stabilizing interactions play a critical role in the manifestation of homotropic cooperativity in aspartate transcarbamoylase by the stabilization of the T state of the enzyme. As substrate binding energy is utilized, more and more of the T state stabilizing interactions are relaxed, and finally the enzyme shifts to the R state. In the case of the Glu-239 stabilizing interactions more than three of the interactions must be broken before the enzyme shifts to the R state. The interactions between the catalytic and regulatory chains and between the two catalytic trimers of aspartate transcarbamoylase provide a global set of interlocking interactions that stabilize the T and R states of the enzyme. The substrate-induced local conformational changes observed in the structure of the isolated catalytic subunit drive the quaternary T to R transition of aspartate transcarbamoylase and functionally induced homotropic cooperativity. Stabilization of the T and R allosteric states ofEscherichia coli aspartate transcarbamoylase is governed by specific intra- and interchain interactions. The six interchain interactions between Glu-239 in one catalytic chain of one catalytic trimer with both Lys-164 and Tyr-165 of a different catalytic chain in the other catalytic trimer have been shown to be involved in the stabilization of the T state. In this study a series of hybrid versions of aspartate transcarbamoylase was studied to determine the minimum number of these Glu-239 interactions necessary to maintain homotropic cooperativity and the T allosteric state. Hybrids with zero, one, and two Glu-239 stabilizing interactions do not exhibit cooperativity, whereas the hybrids with three or more Glu-239 stabilizing interactions exhibit cooperativity. The hybrid enzymes with one or more of the Glu-239 stabilizing interactions also exhibit heterotropic interactions. Two hybrids with three Glu-239 stabilizing interactions, in different geometric relationships, had identical properties. From this and previous studies, it is concluded that the 239 stabilizing interactions play a critical role in the manifestation of homotropic cooperativity in aspartate transcarbamoylase by the stabilization of the T state of the enzyme. As substrate binding energy is utilized, more and more of the T state stabilizing interactions are relaxed, and finally the enzyme shifts to the R state. In the case of the Glu-239 stabilizing interactions more than three of the interactions must be broken before the enzyme shifts to the R state. The interactions between the catalytic and regulatory chains and between the two catalytic trimers of aspartate transcarbamoylase provide a global set of interlocking interactions that stabilize the T and R states of the enzyme. The substrate-induced local conformational changes observed in the structure of the isolated catalytic subunit drive the quaternary T to R transition of aspartate transcarbamoylase and functionally induced homotropic cooperativity. catalytic subunit of aspartate transcarbamoylase composed of three catalytic chains regulatory subunit of aspartate transcarbamoylase composed of two regulatory chains N-(phosphonoacetyl)-l-aspartate the aspartate concentration at half the maximal observed specific activity the catalytic subunit in which each chain has the native Glu at position 239 and has 6 aspartate residues added to the C-terminus (this species is not formed by reconstitution) the mutant catalytic subunit of aspartate transcarbamoylase in which each chain has Glu-239 replaced by Gln (this species is not formed by reconstitution) the reconstituted hybrid catalytic subunit in which two chains have Glu and one chain has Gln at position 239 the reconstituted hybrid catalytic subunit in which one chain has Glu and two chains have Gln at position 239 polyacrylamide gel electrophoresis Allosteric regulation is modulated by molecular transitions within a polymeric enzyme between a low activity low affinity T state and a high activity high affinity R state (1Monod J. Wyman J. Changeux J.P. J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Scopus (6184) Google Scholar). The two states may differ on the quaternary level due to differences in the relative positions of subunits with respect to each other. Interface contacts may undergo significant changes during the T to R state transition, and these contacts often contribute to the relative stabilization of the two states of the enzyme. Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2), which catalyzes the committed step of pyrimidine biosynthesis, the condensation of carbamoyl phosphate and l-aspartate to formN-carbamoyl-l-aspartate and inorganic phosphate (2Gerhart J.C. Pardee A.B. J. Biol. Chem. 1962; 237: 891-896Abstract Full Text PDF PubMed Google Scholar), has become a model system for the study of allosteric regulation. The enzyme shows homotropic cooperativity for the substratel-aspartate and is heterotropically regulated by ATP, CTP (2Gerhart J.C. Pardee A.B. J. Biol. Chem. 1962; 237: 891-896Abstract Full Text PDF PubMed Google Scholar), and UTP in the presence of CTP (3Wild J.R. Loughrey-Chen S.J. Corder T.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 46-50Crossref PubMed Scopus (116) Google Scholar). The holoenzyme from E. coli is a dodecamer composed of six catalytic chains (C)1 ofM r 34,000 and six regulatory chains (R) ofM r 17,000. The catalytic chains are organized as two trimeric subunits, and the regulatory chains are organized as three dimeric subunits. The active sites are located at the interfaces between the catalytic chains, and the nucleotide effectors bind to the same site on each of the regulatory chains (4Gerhart J.C. Pardee A.B. Cold Spring Harbor Symp. Quant. Biol. 1963; 28: 491-496Crossref Google Scholar, 5Ladjimi M.M. Ghellis C. Feller A. Cunin R. Glansdorff N. Pierard A. Hervé G. J. Mol. Biol. 1985; 186: 715-724Crossref PubMed Scopus (37) Google Scholar, 6Changeux J.-P. Gerhart J.C. Schachman H.K. Biochemistry. 1968; 7: 531-538Crossref PubMed Scopus (133) Google Scholar, 7Honzatko R.B. Crawford J.L. Monaco H.L. Ladner J.E. Edwards B.F.P. Evans D.R. Warren S.G. Wiley D.C. Ladner R.C. Lipscomb W.N. J. Mol. Biol. 1982; 160: 219-263Crossref PubMed Scopus (172) Google Scholar, 8Gouaux J.E. Lipscomb W.N. Biochemistry. 1990; 29: 389-402Crossref PubMed Scopus (72) Google Scholar). Two functionally and structurally different states of aspartate transcarbamoylase have been characterized (9Kantrowitz E.R. Lipscomb W.N. Trends Biochem. Sci. 1990; 15: 53-59Abstract Full Text PDF PubMed Scopus (122) Google Scholar). The conversion from the T to R state occurs upon aspartate binding to the enzyme in the presence of carbamoyl phosphate. In addition to quaternary changes, several tertiary changes also occur in the T to R state transition; specifically the 80s and 240s loops of the catalytic chains reorganize. The distinct interchain contacts of side chains of the 240s loop in the T and R states have been identified as being major contributors to stabilization of the T and R states (10Ladjimi M.M. Kantrowitz E.R. Biochemistry. 1988; 27: 276-283Crossref PubMed Scopus (68) Google Scholar). For example, site-specific mutagenesis experiments have identified the interactions of Glu-239 as critical for the stabilization of the T and R states of the enzyme (10Ladjimi M.M. Kantrowitz E.R. Biochemistry. 1988; 27: 276-283Crossref PubMed Scopus (68) Google Scholar). As seen in Fig. 1, Glu-239 of C1 2C followed by a number, e.g. C1 or C4, refers to a particular polypeptide chain in aspartate transcarbamoylase. forms interchain interactions with Lys-164 and Tyr-165 of C4 in the T state (11Krause K.L. Voltz K.W. Lipscomb W.N. J. Mol. Biol. 1987; 193: 527-553Crossref PubMed Scopus (172) Google Scholar). Conversion to the R state breaks these interactions and establishes new intrachain interactions between Glu-239 of C1 and Lys-164 and Tyr-165 of C1. Due to the symmetry of the enzyme, six of these interactions exist within the molecule. Site-specific mutagenesis of Glu-239 to Gln eliminates all six interactions, resulting in a mutant enzyme that functionally exhibits R state kinetics. In the presence of saturating concentrations of carbamoyl phosphate, the mutant enzyme exhibits no homotropic cooperativity and little if any heterotropic interactions. Based upon small-angle x-ray scattering, the mutant enzyme structurally exists in an altered conformation which is shifted significantly toward the R state (12Tauc P. Vachette P. Middleton S.A. Kantrowitz E.R. J. Mol. Biol. 1990; 214: 327-335Crossref PubMed Scopus (25) Google Scholar). The loss of these interchain interactions also substantially reduces the stability of the T state as judged by the ability of saturating concentrations of carbamoyl phosphate, without aspartate, to induce the transition to the R state (10Ladjimi M.M. Kantrowitz E.R. Biochemistry. 1988; 27: 276-283Crossref PubMed Scopus (68) Google Scholar, 13Tauc P. Keiser R.T. Kantrowitz E.R. Vachette P. Protein Sci. 1994; 3: 1998-2004Crossref PubMed Scopus (15) Google Scholar). In order to establish how much stabilization energy is necessary to maintain aspartate transcarbamoylase in the T state, a hybrid enzyme was studied in which one catalytic trimer had the native Glu at position 239 and the other catalytic trimer had Glu-239 replaced by Gln (14Sakash 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). This hybrid enzyme had only three of the six Glu-239 interactions that occur in the wild-type enzyme. The hybrid enzyme exhibited cooperativity and had an aspartate affinity and maximal velocity similar to both parent enzymes. Furthermore, the hybrid enzyme showed an approximately 50% response with the nucleotide effectors. On a functional level, these results suggest that three interactions are sufficient for stabilization of the T state of the enzyme. On a structural level, small-angle x-ray scattering studies showed that the hybrid enzyme is in an alternate conformation close to the T state of the wild-type enzyme. Although carbamoyl phosphate can initiate a slight conformational change toward the R state, the enzyme remains in a T-like state. In this study, we evaluate the contribution that each of the interchain interactions involving Glu-239 has on stabilizing the T and R states of the enzyme, and the contribution that each of these interchain interactions makes to the homotropic and heterotropic properties of the aspartate transcarbamoylase. Agarose, ATP, CTP, l-aspartate,N-carbamoyl-l-aspartate, potassium dihydrogen phosphate, and uracil were obtained from Sigma. Ampicillin, Tris, Q-Sepharose Fast Flow and Source-Q were purchased from Amersham Pharmacia Biotech. UnoQ-1, Protein Assay Dye, and sodium dodecyl sulfate 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 (2Gerhart 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. Antipyrine and sodium pyrophosphate were obtained from Fisher. The E. coli strain MV1190 [Δ(lac-proAB), supE,thi, Δ(sri-recA)306::Tn10(tetr)/F′traD36, proAB, lacI q,lacZΔM15] was obtained from J. Messing. EK1104 [F- ara, thi, Δ(pro-lac), ΔpyrB,pyrF ±, rpsL] was previously constructed in this laboratory (15Nowlan S.F. Kantrowitz E.R. J. Biol. Chem. 1985; 260: 14712-14716Abstract Full Text PDF PubMed Google Scholar). The aspartate tail catalytic subunit (239CEEE) and mutant catalytic subunit (239CQQQ) of aspartate transcarbamoylase were overexpressed utilizing strain EK1104 containing phagemids pEK357 (14Sakash 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) and pEK316 (14Sakash 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), respectively. Bacteria were cultured for 24 h at 37 °C with agitation in M9 media (16Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, 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. After two 65% ammonium sulfate fractionation steps, anion-exchange chromatography using Q-Sepharose Fast Flow resin was used to purify the enzyme further (17Stebbins J.W. Xu W. Kantrowitz E.R. Biochemistry. 1989; 28: 2592-2600Crossref PubMed Scopus (53) Google Scholar). After concentration, the purity of the enzymes was checked by SDS-PAGE (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and nondenaturing PAGE (19Ornstein L. Ann. N. Y. Acad. Sci. 1964; 121: 321-349Crossref PubMed Scopus (3332) Google Scholar, 20Davis B.J. Ann. N. Y. Acad. Sci. 1964; 121: 680-685Google Scholar). The aspartate transcarbamoylase regulatory subunit (R) was overexpressed utilizing strain EK1104 containing phagemid pEK168 (14Sakash 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 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. After two 65% ammonium sulfate fractionation steps, two anion-exchange chromatography steps were utilized to purify the enzyme. The first used Q-Sepharose Fast Flow resin while the second utilized the high resolution, Source-Q resin (14Sakash 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). After concentration of the regulatory subunit, the purity of the enzyme was checked by SDS-PAGE (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and nondenaturing PAGE (19Ornstein L. Ann. N. Y. Acad. Sci. 1964; 121: 321-349Crossref PubMed Scopus (3332) Google Scholar,20Davis B.J. Ann. N. Y. Acad. Sci. 1964; 121: 680-685Google Scholar). Equal amounts of native 239CEEE and native 239CQQQ were mixed and diluted to a final concentration of 7 mg/ml with 50 mmTris-Cl buffer, pH 8.3, 20 mm sodium pyrophosphate, 2 mm 2-mercaptoethanol, and 0.5 mm EDTA, similar to the method previously described (21Lahue R.S. Schachman H.K. J. Biol. Chem. 1984; 259: 13906-13913Abstract Full Text PDF PubMed Google Scholar). This mixture was then dialyzed against 50 mm Tris-Cl buffer, pH 8.3, 20 mmsodium pyrophosphate, 2 mm 2-mercaptoethanol, and 0.5 mm EDTA for 24 h. After equilibrating into 40 mm KH2PO4 buffer, pH 7.0, this mixture was examined by nondenaturing PAGE to confirm the formation of four catalytic subunit species, 239CEEE, 239CEEQ, 239CEQQ, and 239CQQQ. This mixture was fractionated by ion-exchange chromatography using a Bio-Rad Uno Q-1 column. The column was equilibrated with 40 mmKH2PO4 buffer, pH 7.0. After the sample was injected, the column was washed, at a flow rate of 1 ml/min, for 4 min with 40 mm KH2PO4 buffer, pH 7.0, followed by 0–50% 40 mm KH2PO4buffer, pH 7.0, 1.0 m NaCl gradient over 20 min. Nondenaturing PAGE was used to identify the fractions containing 239CEEE, 239CEEQ, 239CEQQ, and 239CQQQ. The fractions were pooled and dialyzed against 40 mm KH2PO4 buffer, pH 7.0. Each species was rechromatographed a second time using identical conditions. Equal amounts of 239CEEQ and 239CEQQ were mixed with excess regulatory subunit and dialyzed overnight against 50 mmTris acetate buffer, pH 8.3, 2 mm 2-mercaptoethanol, and 0.1 mm zinc acetate (22Sakash 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, (239CEEQ)2R3, (239CEEQ)(239CEQQ)R3, and (239CEQQ)2R3. Separation of the species was performed using the UNO Q-1 column as indicated previously. Equal amounts of 239CEQQ and 239CQQQ were mixed with excess regulatory subunit and dialyzed overnight against 50 mm Tris acetate buffer, pH 8.3, 2 mm2-mercaptoethanol, and 0.1 mm zinc acetate. The mixture was examined by nondenaturing PAGE to confirm the existence of the three holoenzyme species, (239EQQ)2R3, (239EQQ)(239QQQ)R3, and (239QQQ)2R3. Separation of the species was performed using the UNO Q-1 column as indicated previously. The concentration of the wild-type holoenzyme, catalytic subunit, and regulatory subunit were determined from absorbance measurements at 280 nm using extinction coefficients of 0.59, 0.72, and 0.32 cm2mg−1, respectively (23Gerhart J.C. Holoubek H. J. Biol. Chem. 1967; 242: 2886-2892Abstract Full Text PDF PubMed Google Scholar, 24Dembowski N.J. Kantrowitz E.R. Protein Eng. 1993; 6: 123-127Crossref PubMed Scopus (7) Google Scholar). The concentrations of the mutant enzymes were determined by the Bio-Rad version of the Bradford dye-binding assay (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). The aspartate transcarbamoylase activity was measure at 25 °C by the colorimetric method (26Pastra-Landis S.C. Foote J. Kantrowitz E.R. Anal. Biochem. 1981; 118: 358-363Crossref PubMed Scopus (111) 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 (27Silver R.S. Daigneault J.P. Teague P.D. Kantrowitz E.R. J. Mol. Biol. 1983; 168: 729-745Crossref PubMed Scopus (45) Google Scholar). As has been previously shown (21Lahue R.S. Schachman H.K. J. Biol. Chem. 1984; 259: 13906-13913Abstract Full Text PDF PubMed Google Scholar), interchange of catalytic chains occurs when the catalytic subunit of aspartate transcarbamoylase is treated with sodium pyrophosphate. The intrachain catalytic subunit hybrids 239CEEQ and 239CEQQ were generated by mixing native 239CQQQwith native 239CEEE and then dialyzing the mixture against buffer containing 20 mm sodium pyrophosphate. The four species, 239CEEE, 239CEEQ, 239CEQQ, and 239CQQQ, were identified by nondenaturing PAGE and purified by anion-exchange chromatography (see Fig.2), The six aspartate residues attached to each catalytic chain containing Glu-239 give a sufficient difference in charge for the four catalytic subunit species to be eluted differentially by a salt gradient on the anion-exchange column (see Fig. 2). In previous work (14Sakash 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), the symmetric hybrid holoenzyme (239CEEE)(239CQQQ)R3, in which one catalytic trimer had the mutation at position 239, was investigated. This hybrid holoenzyme exhibited cooperativity and nucleotide effects that were not observed for the (239CEEE)2R3 holoenzyme. In this work the asymmetric (239CEEQ)(239CEQQ)R3holoenzyme was investigated. This enzyme has the same number of 239 interchain interactions as the symmetric hybrid (239CEEE)(239CQQQ)R3 but in a different geometric arrangement. The hybrid (239CEEQ)(239CEQQ)R3holoenzyme was generated by mixing 239CEEQ and 239CEQQ and regulatory subunit in excess. The species were identified by nondenaturing PAGE, and the (239CEEQ)(239CEQQ)R3 holoenzyme was purified by anion-exchange chromatography. Fig.3 shows the aspartate saturation curves produced by the reconstituted mutant enzymes, and the kinetic parameters calculated from these curves are given in TableI. The (239CEEQ)(239CEQQ)R3 holoenzyme exhibited a sigmoidal saturation curve with a Hill coefficient of 1.4 similar to the saturation curve of the symmetric (239CEEE)(239CQQQ)R3 holoenzyme previously characterized (14Sakash 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). The maximal velocity of the (239CEEQ)(239CEQQ)R3 holoenzyme was 16.0 mmol/h mg and the [Asp]0.5 was 6.6 mm, both of which are similar to the parameters of the symmetric (239CEEE)(239CQQQ)R3holoenzyme.Table IKinetic parameters of E239Q hybrid holoenzymes at pH 8.3EnzymeNo. of 239 interactionsMaximal velocity1-aMaximal velocity represents the maximal observed specific activity from the aspartate saturation curves.[Asp]0.5n Hmmol · mg −1 · h −1mm(239CEEE)2R3619.3 ± 1.81-bData from Sakash et al.(14).9.6 ± 0.82.6 ± 0.5(239CEEQ)2R3417.5 ± 1.21-cAverage deviation of three determinations.7.0 ± 0.21.6 ± 0.09(239CEEQ)(239CEQQ)R3316.0 ± 2.66.6 ± 0.71.4 ± 0.03(239CEEE)(239CQQQ)R3321.2 ± 1.81-bData from Sakash et al.(14).6.4 ± 0.21.4 ± 0.09EnzymeNo. of 239 interactionsMaximal velocityK mn Hmmol · mg −1 · h −1mm(239CEQQ)(239CEQQ)R3218.0 ± 0.85.4 ± 0.41(239CEQQ)(239CQQQ)R3116.2 ± 0.65.6 ± 0.091(239CQQQ)2R31-bData from Sakash et al.(14).020.0 ± 0.68.4 ± 0.21These data were determined from the aspartate saturation curves. Colorimetric assays were performed at 25 °C in 50 mmTris acetate buffer, pH 8.3, and saturating levels of carbamoyl phosphate (4.8 mm).1-a Maximal velocity represents the maximal observed specific activity from the aspartate saturation curves.1-b Data from Sakash et al.(14Sakash 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).1-c Average deviation of three determinations. Open table in a new tab These data were determined from the aspartate saturation curves. Colorimetric assays were performed at 25 °C in 50 mmTris acetate buffer, pH 8.3, and saturating levels of carbamoyl phosphate (4.8 mm). The influence of the allosteric effectors ATP and CTP on the asymmetric (239CEEQ)(239CEQQ)R3 holoenzyme was determined at one-half the [Asp]0.5 (see Fig.4). This concentration of aspartate was selected since the nucleotides exert a larger influence on the activity of the enzyme as the aspartate concentration is reduced (28Tauc 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 (Table II). As seen in Fig. 4, ATP activates the (239CEEQ)(239CEQQ)R3 holoenzyme 254% and CTP inhibits to a residual activity 34%, which are essentially the same as the values determined for the symmetric (239CEEE)(239CQQQ)R3 holoenzyme (14Sakash 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).Table IICTP inhibition and ATP activation of the mutant holoenzymes at subsaturating aspartate at pH 8.3EnzymeCTP parametersATP parametersResidual activity2-aPercent residual activity is defined as 100 (A CTP)/A whereA CTP is the activity in the presence of CTP andA is the activity in the absence of CTP.K CTP2-bK is the nucleotide concentration required to activate or inhibit the enzyme by 50% of the maximal effect.% Activation2-cPercent activation is defined as 100 (AATP)/A, whereAATP is the activity in the presence of ATP andA is the activity in the absence of ATP.K ATP2-bK is the nucleotide concentration required to activate or inhibit the enzyme by 50% of the maximal effect.%mmmm(239CEEE)2R319.5 ± 1.6 d,e0.08 ± 0.04504 ± 800.6 ± 0.2(239CEEQ)2R329.3 ± 3.90.08 ± 0.04345 ± 330.6 ± 0.2(239CEEQ)(239CEQQ)R333.7 ± 4.50.05 ± 0.03254 ± 220.4 ± 0.1(239CEEE)(239CQQQ)R336.9 ± 0.32-eData from Sakash et al.(14).0.1 ± 0.01258 ± 130.4 ± 0.07(239CEQQ)2R346.5 ± 7.10.1 ± 0.03147 ± 70.2 ± 0.1(239CEQQ)(239CQQQ)R367.4 ± 2.70.3 ± 0.1110 ± 10.3 ± 0.2(239CQQQ)2R32-dAverage deviation of three determinations.90.0 ± 7.02-eData from Sakash et al.(14).ND2-fND, not determined.NoneND2-fND, not determined.These data were determined from ATP and CTP saturation curves (Fig. 4). Colorimetric assays were performed at 25 °C in 50 mmTris acetate buffer, pH 8.3. ATP and CTP saturation curves were determined at saturating levels of carbamoyl phosphate (4.8 mm) and aspartate concentrations at one-half the [Asp]0.5 of the respective holoenzyme at pH 8.3.2-a Percent residual activity is defined as 100 (A CTP)/A whereA CTP is the activity in the presence of CTP andA is the activity in the absence of CTP.2-b K is the nucleotide concentration required to activate or inhibit the enzyme by 50% of the maximal effect.2-c Percent activation is defined as 100 (AATP)/A, whereAATP is the activity in the presence of ATP andA is the activity in the absence of ATP.2-d Average deviation of three determinations.2-e Data from Sakash et al.(14Sakash 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).2-f ND, not determined. Open table in a new tab These data were determined from ATP and CTP saturation curves (Fig. 4). Colorimetric assays were performed at 25 °C in 50 mmTris acetate buffer, pH 8.3. ATP and CTP saturation curves were determined at saturating levels of carbamoyl phosphate (4.8 mm) and aspartate concentrations at one-half the [Asp]0.5 of the respective holoenzyme at pH 8.3. The hybrid holoenzymes containing two out of six mutations at position 239, (239CEEQ)2R3, and four out of six mutations at position 239, (239QEQQ)2R3, were formed by mixing 239CEEQ, 239CEQQ, and regulatory subunit in excess. Three species were identified by nondenaturing PAGE and purified by anion-exchange chromatography. In a similar fashion, the hybrid holoenzyme containing one out of six mutations at position 239, (239CEQQ)(239CQQQ)R3, was formed by reconstitution of 239CEQQ and 239CQQQ with excess regulatory subunit followed by chromatography as indicated above. Fig. 3 shows the aspartate saturation curves produced by the reconstituted mutant enzymes, and the kinetic parameters calculated from these curves are shown in Table I. The (239CEEQ)2R3 holoenzyme exhibits a sigmoidal saturation curve with a Hill coefficient of 1.6. The maximal velocity of the (239CEEQ)2R3holoenzyme was 17.5 mmol/h mg, and the [Asp]0.5 was 7.0 mm (Table I). On the other hand, the (239CEQQ)2R3 and (239CEQQ)(239CQQQ)R3 holoenzymes exhibit hyperbolic saturation curves. The (239CEQQ)2R3 holoenzyme has an observed maximal specific activity of 18.0 mmol/mg h and aK m of 5.4 mm; the (239CEQQ)(239CQQQ)R3 holoenzyme has an observed maximal specific activity of 16.2 mmol/mg h and aK m of 5.6 mm. Nucleotide saturation curves with CTP and ATP were determined with the (239CEEQ)2R3, (239CEQQ)2R3, and (239CEQQ)(239CQQQ)R3 holoenzymes at one-half the [Asp]0.5 (Fig. 4). As seen in Fig. 4, ATP activates and CTP inhibits all three holoenzymes. The (239CEEQ)2R3 holoenzyme is activated by ATP 345% and inhibited by CTP to a residual activity of 29.3%; these parameters indicate a greater response than the (239CEEQ)(239CEQQ)R3 holoenzyme but a reduced response compared with the (239EEE)2R3 holoenzyme. The (239CEQQ)2R3 holoenzyme is activated by ATP 147%, and the (239CEQQ)(239CQQQ)R3 holoenzyme is activated 110%. These enzymes are inhibited by CTP to a residual activity of 46.5 and 67.4%, respectively. The bisubstrate analog PALA is able to activate wild-type aspartate transcarbamoylase holoenzyme at low concentrations of aspartate and saturating concentrations of carbamoyl phosphate. This activation is due to the ability of this compound to convert the enzyme from the low activity, low affinity T state to the high activity, high affinity R state. This activation can therefore be used to verify the ability of a mutant enzyme to undergo the allosteric transition. Therefore, the effect of PALA on the mutant intrasubunit hybrid enzymes was determined at 0.2× [Asp]0.5 (data not shown). PALA activated both (239CEEQ)2R3 and (239CEEQ)(239CEQQ)R3 holoenzymes; the (239CEEQ)2R3 holoenzyme was activated to a slightly greater extent than (239CEEQ)(239CEQQ)R3 holoenzyme. PALA inhibited both the (239CEQQ)2R3 and (239CEQQ)(239CQQQ)R3 holoenzymes, with no detectable initial activation. The ability of E. coli aspartate transcarbamoylase to regulate pyrimidine biosynthesis is due to its ability to alter its activity in both a homotropic and heterotropic fashion. A characteristic of allosteric enzymes is that they exist in two or more functional and structural states that differ in activity and affinity. For aspartate transcarbamoylase these two states (T and R) are structurally different, and each structure is stabilized by different interchain and interdomain interactions. Many of these stabilizing interactions have been characterized (9Kantrowitz E.R. Lipscomb W.N. Trends Biochem. Sci. 1990; 15: 53-59Abstract Full Text PDF PubMed Scopus (122) Google Scholar), and one of the most important interactions for establishment of both the homotropic cooperativity and heterotropic interactions involves Glu-239 of the catalytic chain. Previous studies have shown that the replacement of Glu-239 with Gln in all six catalytic chains of aspartate transcarbamoylase results in a holoenzyme devoid of both homotropic and heterotropic interactions (10Ladjimi M.M. Kantrowitz E.R. Biochemistry. 1988; 27: 276-283Crossref PubMed Scopus (68) Google Scholar). However, when only three of the six 239 positions are replaced by Gln both homotropic and heterotropic interactions are restored (14Sakash 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). As seen in Fig. 1, Glu-239 is involved in intersubunit interactions in the T state and intrachain interactions in the R state. Small-angle x-ray scattering experiments have shown that when all six of the Glu-239 interactions are broken, the mutant enzyme exists in a new quaternary structure, different from either T or R, but shifted toward the R state of the wild-type enzyme (12Tauc P. Vachette P. Middleton S.A. Kantrowitz E.R. J. Mol. Biol. 1990; 214: 327-335Crossref PubMed Scopus (25) Google Scholar). The fact that this scattering pattern is shifted completely to the R state in the presence of only carbamoyl phosphate explains the lack of both homotropic and heterotropic interactions in the mutant enzyme. However, when only three of the Glu-239 interactions are broken, small-angle x-ray scattering data suggest an enzyme that remains essentially in the T state. This explains the restoration of both homotropic and heterotropic interactions (14Sakash 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). We have previously shown that the addition of six aspartic acid residues as a C-terminal extension of the catalytic chain of aspartate transcarbamoylase is an effective method for the preparation of hybrid versions of aspartate transcarbamoylase in which the two catalytic subunits are different (14Sakash 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). Here we show that this same Asp tail extension is also effective for preparation of hybrid versions of the catalytic subunit, in which one or more of the chains are different. By using this methodology, we have been able to prepare catalytic subunits in which one or two chains have Glu-239 replaced by Gln. These intrachain hybrid catalytic subunits were then used to create hybrid holoenzymes that have a varying number of Glu-239 stabilizing interactions. With the holoenzymes containing one or two Glu-239 interactions per molecule, we were able to evaluate the minimal number of interactions necessary to stabilize the T state of aspartate transcarbamoylase and therefore allow the enzyme to exhibit an allosteric transition. Other hybrids have allowed us to determine if the interactions between the upper and lower catalytic trimers of aspartate transcarbamoylase must be in a particular geometric arrangement for function. Both the previously investigated symmetric hybrid holoenzyme, (239CEEE)(239CQQQ)R3 (14Sakash 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), and the asymmetric hybrid holoenzyme, (239CEEQ)(239CEQQ)R3, reported here, have three of the six possible Glu-239 interchain-stabilizing interactions. However, in the asymmetric hybrid holoenzyme two interactions originate from one catalytic trimer and one interaction originates from the other catalytic trimer. The asymmetric hybrid holoenzyme exhibits both homotropic and heterotropic properties that are very similar to the symmetric hybrid holoenzyme (Table I and TableII). Thus, the arrangement of the three stabilizing interactions (i.e. symmetric versus asymmetric) does not influence the homotropic and heterotropic properties of the enzyme. These data suggest that the number of stabilizing interactions and not the geometric arrangement of the interactions affects the global stabilization of the enzyme and is the critical requirement for the manifestation of homotropic and heterotropic interactions in aspartate transcarbamoylase. The cooperativity of aspartate transcarbamoylase is related to the number of Glu-239 stabilizing interactions. As seen in Fig.5, the hybrids that have less than three Glu-239 stabilizing interactions do not exhibit homotropic cooperativity. These results suggest that less than three interactions across the trimer interface do not provide sufficient stabilization to maintain the enzyme in the T state, and therefore homotropic cooperativity is lost. However, all the hybrid enzymes that have three or more of the Glu-239 stabilizing interactions exhibit cooperativity. Furthermore, the extent of cooperativity observed with these hybrid enzymes is enhanced as the number of the Glu-239 stabilizing interactions increases (see Fig. 5 and Table I). These functional data together with structural data from small-angle x-ray scattering (14Sakash 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) indicate that three Glu-239 stabilizing interactions are sufficient to stabilize the enzyme in the T state allowing both a functional and structural transition to the R state. When one additional Glu-239 stabilizing interaction is eliminated cooperativity is destroyed. Thus, the difference in free energy between an enzyme with and without homotropic cooperativity is essentially one salt link. Each of the Glu-239 interactions adds to the global stabilization of the T state of the enzyme. As links are lost, the T state is progressively destabilized until the fourth is eliminated. With only two Glu-239 stabilizing interactions, the T state quaternary structure is no longer stable, and the enzyme shifts toward an R-like conformation. The addition of carbamoyl phosphate may convert the enzyme to a state nearly identical to the R state of the wild-type enzyme as was observed for the (239CEEE)2R3 holoenzyme (12Tauc P. Vachette P. Middleton S.A. Kantrowitz E.R. J. Mol. Biol. 1990; 214: 327-335Crossref PubMed Scopus (25) Google Scholar). Contrary to the results describing homotropic cooperativity, the heterotropic interactions, such as the extent of ATP activation and CTP inhibition, are related to the number of Glu-239 stabilizing interactions (see Fig. 5). Furthermore, the extent of the response to both ATP and CTP is related to whether or not the enzyme exhibits homotropic cooperativity. As seen in Fig. 5, CTP has a strong inhibitory effect on the hybrid enzymes that have only one or two Glu-239 stabilizing interactions. With only one or two Glu-239 stabilizing interactions, the enzyme exists in an R-like state in which CTP can function both to shift the allosteric equilibrium back to the T state and to have a direct inhibitory influence on catalysis. However, wild-type like CTP inhibition is not observed for these hybrids suggesting that the Glu-239 interactions must be necessary to fully stabilize the enzyme in the T state. The ability of the nucleotides to function by both a primary and secondary mode has been previously proposed (28Tauc P. Leconte C. Kerbiriou D. Thiry L. Hervé G. J. Mol. Biol. 1982; 155: 155-168Crossref PubMed Scopus (48) Google Scholar). In the absence of cooperativity, ATP is only able to activate these hybrids slightly, suggesting that ATP is unable to shift the enzyme toward the R state, since the quaternary structure of the holoenzyme is essentially R. The hybrid enzymes that demonstrate cooperativity, with three or more Glu-239 stabilizing interactions, have a dramatically different response toward the nucleotides. As the number of Glu-239 stabilizing interactions increases, the additional inhibition by CTP decreases, and the additional activation by ATP increases (see Fig. 5). In the case of ATP, the establishment of cooperativity in the hybrids with three or more Glu-239 stabilizing interactions makes the R state more accessible, and ATP shows significantly enhanced stimulation. All six Glu-239 interactions are necessary for a fully T state holoenzyme. Even the presence of CTP does not add sufficient stabilization for the holoenzyme to attain the T state if one Glu-239 interaction is broken. The binding energy of CTP can not compensate for the energy lost by the removal of one Glu-239 salt link. The effect of missing Glu-239 salt links is globally distributed throughout the holoenzyme based upon the kinetic and nucleotide studies on this series of holoenzymes. Each salt link may act as a clamp, holding the holoenzyme in the T conformation. When the first and second salt links are “systematically” removed, it is as if the clamps have been removed and the enzyme flips toward the R state as indicated by the sharp initial decrease in the ATP effect on the one and two mutant hybrid enzymes. CTP may be able to shift the holoenzyme back toward the T state; however, without the salt links, the enzyme cannot be clamped down in the T state. From this and previous studies (10Ladjimi M.M. Kantrowitz E.R. Biochemistry. 1988; 27: 276-283Crossref PubMed Scopus (68) Google Scholar, 12Tauc P. Vachette P. Middleton S.A. Kantrowitz E.R. J. Mol. Biol. 1990; 214: 327-335Crossref PubMed Scopus (25) Google Scholar, 14Sakash 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), it is clear that the 239 stabilizing interactions play a critical role in the manifestation of homotropic cooperativity in aspartate transcarbamoylase. The interchain interactions involving Glu-239 are critically important for stabilization of the T state. However, the binding of aspartate, after the ordered binding of carbamoyl phosphate, to one or so of the active sites initiates the global conformational change to the R state. The energy associated with aspartate binding induces the closure of the aspartate and carbamoyl phosphate domains of the particular catalytic chain to which the aspartate bound. This domain closure induces alterations in the 80s loop of an adjacent catalytic chain necessary to complete the formation of the active site as well as a repositioning of the 240s loop that allows a set of intradomain bridging interactions involving Arg-229, Arg-233, and Glu-50 to stabilize the domain-closed high activity, high affinity active site. The closure of these domains and the movement of the 240s loop influence the Glu-239 interchain-stabilizing interactions in two ways. First, as a 240s loop repositions, the corresponding Glu-239 interchain link becomes destabilized and is broken. Second, this 240s loop cannot adapt its R state conformation without the quaternary conformational change of the molecule to relieve the steric clash of this 240s loop with a nearby 240s loop moving in the opposite direction (10Ladjimi M.M. Kantrowitz E.R. Biochemistry. 1988; 27: 276-283Crossref PubMed Scopus (68) Google Scholar). As substrate-binding energy is utilized, more and more of the T state stabilizing interactions are relaxed and finally the enzyme flips to the R state. In the case of the Glu-239 stabilizing interactions, more than three of these interactions must be broken before the enzymes shifts to the R state. It has recently been suggested (29Endrizzi J.A. Beernink P.T. Alber T. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5077-5082Crossref PubMed Scopus (35) Google Scholar) that the similar structure between the isolated catalytic subunit in the unligated and PALA-ligated states compared with the catalytic subunit of the unligated and PALA-ligated states of the holoenzyme indicates that the conformational differences observed between the unligated and ligated states of the catalytic subunit are not responsible for homotropic cooperativity. However, when the catalytic and regulatory subunits combine to form the holoenzyme, additional intersubunit interactions are established. This exquisite set of intersubunit interactions between the catalytic and regulatory chains and between the two catalytic trimers, such as the Glu-239 stabilizing interactions, provides a global set of interlocking interactions for the holoenzyme. These interactions work in a concerted fashion with the substrate-induced local conformational changes, as observed in the structure of the isolated catalytic subunit, to drive the quaternary T to R transition of aspartate transcarbamoylase and functionally induced homotropic cooperativity. We thank Robin S. Chan and Tatyana Vorobyova for help in purifying the large quantities of the catalytic and regulatory subunits necessary for these experiments.