FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1968313869> ?p ?o ?g. }

• W1968313869 endingPage "23310" @default.
• W1968313869 startingPage "23302" @default.
• W1968313869 abstract "Here the functional and structural importance of interactions involving the 240s loop of the catalytic chain for the stabilization of the T state of aspartate transcarbamoylase were tested by replacement of Lys-244 with Asn and Ala. For the K244A and K244N mutant enzymes, the aspartate concentration required to achieve half-maximal specific activity was reduced to 8.4 and 4.0 mm, respectively, as compared with 12.4 mm for the wild-type enzyme. Both mutant enzymes exhibited dramatic reductions in homotropic cooperativity and the ability of the heterotropic effectors to modulate activity. Small angle x-ray scattering studies showed that the unligated structure of the mutant enzymes, and the structure of the mutant enzymes ligated with N-phosphonacetyl-l-aspartate, were similar to that observed for the unligated and N-phosphonacetyl-l-aspartateligated wild-type enzyme. A saturating concentration of carbamoyl phosphate alone has little influence on the small angle x-ray scattering of the wild-type enzyme. However, carbamoyl phosphate was able to shift the structure of the two mutant enzymes dramatically toward R, establishing that the mutations had destabilized the T state of the enzyme. The x-ray crystal structure of K244N enzyme showed that numerous local T state stabilizing interactions involving 240s loop residues were lost. Furthermore, the structure established that the mutation induced additional alterations at the subunit interfaces, the active site, the relative position of the domains of the catalytic chains, and the allosteric domain of the regulatory chains. Most of these changes reflect motions toward the R state structure. However, the K244N mutation alone only changes local conformations of the enzyme to an R-like structure, without triggering the quaternary structural transition. These results suggest that loss of cooperativity and reduction in heterotropic effects is due to the dramatic destabilization of the T state of the enzyme by this mutation in the 240s loop of the catalytic chain. Here the functional and structural importance of interactions involving the 240s loop of the catalytic chain for the stabilization of the T state of aspartate transcarbamoylase were tested by replacement of Lys-244 with Asn and Ala. For the K244A and K244N mutant enzymes, the aspartate concentration required to achieve half-maximal specific activity was reduced to 8.4 and 4.0 mm, respectively, as compared with 12.4 mm for the wild-type enzyme. Both mutant enzymes exhibited dramatic reductions in homotropic cooperativity and the ability of the heterotropic effectors to modulate activity. Small angle x-ray scattering studies showed that the unligated structure of the mutant enzymes, and the structure of the mutant enzymes ligated with N-phosphonacetyl-l-aspartate, were similar to that observed for the unligated and N-phosphonacetyl-l-aspartateligated wild-type enzyme. A saturating concentration of carbamoyl phosphate alone has little influence on the small angle x-ray scattering of the wild-type enzyme. However, carbamoyl phosphate was able to shift the structure of the two mutant enzymes dramatically toward R, establishing that the mutations had destabilized the T state of the enzyme. The x-ray crystal structure of K244N enzyme showed that numerous local T state stabilizing interactions involving 240s loop residues were lost. Furthermore, the structure established that the mutation induced additional alterations at the subunit interfaces, the active site, the relative position of the domains of the catalytic chains, and the allosteric domain of the regulatory chains. Most of these changes reflect motions toward the R state structure. However, the K244N mutation alone only changes local conformations of the enzyme to an R-like structure, without triggering the quaternary structural transition. These results suggest that loss of cooperativity and reduction in heterotropic effects is due to the dramatic destabilization of the T state of the enzyme by this mutation in the 240s loop of the catalytic chain. Regulation of metabolic pathways via enzymes whose activity is dependent upon the concentration of various metabolites is of paramount importance for all living systems. As a response to a change in the concentration of certain metabolites, a regulatory enzyme can alter its catalytic activity thereby regulating the flux of metabolic intermediates. In particular, a change in function (such as the affinity for substrate) necessarily mandates a change in structure (1Koshland D.E. Nemethy G. Filmer D. Biochemistry. 1966; 5: 365-385Crossref PubMed Scopus (2206) Google Scholar). One excellent example of a regulatory enzyme in which there are dramatic functional and structural changes dependent upon the concentrations of substrates and allosteric effectors is Escherichia coli aspartate transcarbamoylase. Aspartate transcarbamoylase (EC 2.1.3.2) catalyzes the first reaction in pyrimidine biosynthesis, the reaction between carbamoyl phosphate and l-aspartate to form N-carbamoyl-l-aspartate and inorganic phosphate. The enzyme shows homotropic cooperativity for the substrate l-aspartate (2Gerhart J.C. Schachman H.K. Biochemistry. 1968; 7: 538-552Crossref PubMed Scopus (182) Google Scholar). Its activity is inhibited heterotropically by CTP (3Yates R.A. Pardee A.B. J. Biol. Chem. 1956; 221: 757-770Abstract Full Text PDF PubMed Google Scholar), by UTP in the presence of CTP (4Wild 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), and activated heterotropically by ATP (5Bethell M.R. Smith K.E. White J.S. Jones M.E. Proc. Natl. Acad. Sci. U. S. A. 1968; 60: 1442-1449Crossref PubMed Scopus (103) Google Scholar, 6Gerhart J.C. Pardee A.B. J. Biol. Chem. 1962; 237: 891-896Abstract Full Text PDF PubMed Google Scholar, 7Gerhart J.C. Pardee A.B. Cold Spring Harbor Symp. Quant. Biol. 1963; 28: 491-496Crossref Google Scholar), the product of the parallel purine biosynthetic pathway. The holoenzyme consists of two trimeric catalytic subunits (Mr 34,000/chain) and three dimeric regulatory subunits (Mr 17,000/chain). Each of the catalytic subunits contains three active sites shared across the interface between two adjacent catalytic chains (8Krause K.L. Voltz K.W. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1643-1647Crossref PubMed Scopus (86) Google Scholar, 9Robey E.A. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 361-365Crossref PubMed Scopus (65) Google Scholar). The catalytic subunit catalyzes the reaction, whereas the regulatory subunit binds the allosteric effectors and is catalytically inactive (10Gerhart J.C. Schachman H.K. Biochemistry. 1965; 4: 1054-1062Crossref PubMed Scopus (255) Google Scholar). When substrates or suitable substrate analogues bind, the enzyme undergoes a transition from a low-activity low affinity conformation (T state) to a high activity high affinity conformation (R state). During the allosteric transition, the two catalytic subunits separate by 11 Å along the 3-fold axis (vertical expansion) and rotate ∼15° about the 3-fold axis, while the three regulatory subunits rotate ∼15° about their respective 2-fold axes. In addition, the two folding domains of each catalytic chain, the CP 1The abbreviations used are: CP domain, carbamoyl phosphate binding domain of the catalytic chain; c, catalytic chain; r, regulatory chain; ASP domain, aspartate binding domain of the catalytic chain; AL domain, allosteric domain of the regulatory chain; ZN domain, zinc domain of the regulatory chain; SAXS, small angle X-ray scattering; PEG, polyethylene glycol; r.m.s., root mean square; PALA, N-(phosphonoacetyl)-l-aspartate; PDB, Protein Data Bank. and ASP domains, close about the substrates to form the catalytically competent active site, and several subunit interfaces are restructured (11Lipscomb W.N. Adv. Enzymol. 1994; 68: 67-151PubMed Google Scholar). An important functional consequence of this structural reorganization is to increase the affinity of the enzyme for aspartate and to significantly enhance catalytic activity. Although most of the structural rearrangements can be described by rigid body rotations and translations of the two domains of the catalytic chain, the loop composed of residues 73–88 in the CP domain (80′s loop) and residues 230–245 in the ASP domain (240s loop) undergo dramatic conformational changes in the T to R transition that cannot be explained as rigid body domain motions. When the 240s loop reorients it moves toward the CP domain helping to create the high activity high affinity active site (12Ladjimi M.M. Kantrowitz E.R. Biochemistry. 1988; 27: 276-283Crossref PubMed Scopus (68) Google Scholar). The position of the 240s loop in the T and R states are stabilized by a series of salt links and hydrogen bonds. In the T state, these interactions seem to not only position the 240s loop, but also stabilize the compact T structure. When some of these interactions have been disrupted, by amino acid substitution, the T state is destabilized relative to the R state (12Ladjimi M.M. Kantrowitz E.R. Biochemistry. 1988; 27: 276-283Crossref PubMed Scopus (68) Google Scholar, 13Middleton S.A. Kantrowitz E.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5866-5870Crossref PubMed Scopus (34) Google Scholar, 14Middleton S.A. Kantrowitz E.R. Biochemistry. 1988; 27: 8653-8660Crossref PubMed Scopus (31) Google Scholar, 15Middleton S.A. Stebbins J.W. Kantrowitz E.R. Biochemistry. 1989; 28: 1617-1626Crossref PubMed Scopus (29) Google Scholar, 16Ladjimi M.M. Middleton S.A. Kelleher K.S. Kantrowitz E.R. Biochemistry. 1988; 27: 268-276Crossref PubMed Scopus (57) Google Scholar). The exact position of the 240s loop has been difficult to discern because of relatively weak electron density in this portion of the structure (17Kim K.H. Pan Z. Honzatko R.B. Ke H.-M. Lipscomb W.N. J. Mol. Biol. 1987; 196: 853-875Crossref PubMed Scopus (115) Google Scholar, 18Ke H.-M. Honzatko R.B. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4027-4040Crossref Scopus (87) Google Scholar, 19Kosman R.P. Gouaux J.E. Lipscomb W.N. Proteins: Struct. Funct. Genet. 1993; 15: 147-176Crossref PubMed Scopus (66) Google Scholar). In a mutant structure more recently determined (20Williams M.K. Stec B. Kantrowitz E.R. J. Mol. Biol. 1998; 281: 121-134Crossref PubMed Scopus (18) Google Scholar), the quality of the electron density map of the 240s loop was sufficiently high to allow for the conformation of the loop to be more firmly established, including the presence of a short segment of well defined 310 helix at the end of the fragment. In addition, this revised conformation of the 240s loop shows a salt link between Lys-244 and Asp-271 that previously had not been observed. Here, we investigate the role of this interaction for the stabilization of the T state of aspartate transcarbamoylase by site-specific mutagenesis, kinetics, small angle x-ray scattering, and x-ray crystallography. Materials—Agar, l-aspartate, ampicillin, ATP, CTP, carbamoyl phosphate, N-carbamoyl-l-aspartate, 2-mercaptoethanol, polyethylene glycol 1450, potassium dihydrogen phosphate, Bis-Tris, malonic acid, sodium EDTA, sodium acetate, sodium azide, and uracil were obtained from Sigma. Q-Sepharose Fast Flow resin was obtained from Amersham Biosciences. The QuikChange site-directed mutagenesis kit was obtained from Stratagene (La Jolla, CA). Primers were supplied from Operon Technology (Alameda, CA). The QIAprep Miniprep kit from Qiagen (Valencia, CA) was used for plasmid preparation. Casamino acids, yeast extract and tryptone were obtained from Difco (Detroit, MI). Sodium dodecyl sulfate and protein assay dye were purchased from Bio-Rad. Enzyme grade ammonium sulfate, Tris, electrophoresis grade acrylamide, and agarose were purchased from ICN Biomedicals (Costa Mesa, CA). Carbamoyl phosphate dilithium salt, obtained from Sigma, was purified before use by precipitation from 50%(v/v) ethanol and was stored dessicated at -20 °C. Antipyrine was obtained from Fisher Chemical Co. Crystal cryo-mounting loops were obtained from Hampton Research (Laguna Niguel, CA). Construction of Mutant Aspartate Transcarbamoylases—The mutant versions of E. coli aspartate transcarbamoylase were constructed by introducing site-specific base mutations in the pyrB gene using the QuikChange site-directed mutagenesis kit. DNA primers were designed to insert the mutations in both the forward and reverse directions. Site-specific mutagenesis was performed using plasmid pEK152 (21Baker D.P. Kantrowitz E.R. Biochemistry. 1993; 32: 10150-10158Crossref PubMed Scopus (29) Google Scholar) as the template DNA. Candidates were screened for the mutation by DNA sequence analysis. Once candidates were identified, the entire pyrB gene was sequenced to verify that the only mutation was at the 244 codon. The final plasmids with the K244N and K244A mutations were identified as pEK582 and pEK584 respectively. Enzyme Overexpression and Purification—Plasmids pEK152, pEK582, and pEK584 for the wild-type, K244N, and K244A enzymes, respectively, were transformed into the E. coli strain EK1104 for expression and purification as previously described (22Nowlan S.F. Kantrowitz E.R. J. Biol. Chem. 1985; 260: 14712-14716Abstract Full Text PDF PubMed Google Scholar). The enzyme purity was judged both by native PAGE (23Davis B.J. Ann. N. Y. Acad. Sci. 1964; 121: 680-685Google Scholar, 24Ornstein L. Ann. N. Y. Acad. Sci. 1964; 121: 321-349Crossref PubMed Scopus (3332) Google Scholar); as well as by SDS-PAGE (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207479) Google Scholar) stained with Coomassie Blue. Protein Determination—The concentration of pure wild-type enzyme was determined by absorbance measurements at 280 nm with an extinction coefficient of 0.59 cm2·mg-1 (26Gerhart J.C. Holoubek H. J. Biol. Chem. 1967; 242: 2886-2892Abstract Full Text PDF PubMed Google Scholar). The protein concentration of the mutant enzymes was determined by the Bio-Rad version of the Bradford's dye binding assay using the wild-type enzyme as standard (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217390) Google Scholar). Aspartate Transcarbamoylase Assay—The activity of the enzyme was measured at 25 °C by the colorimetric method (28Pastra-Landis S.C. Foote J. Kantrowitz E.R. Anal. Biochem. 1981; 118: 358-363Crossref PubMed Scopus (111) Google Scholar). Saturation kinetics was performed in duplicate, and data points shown are the average values. Assays were performed in 50 mm Tris acetate buffer, pH 8.3 in the presence of a saturating concentration of carbamoyl phosphate (4.8 mm). Analysis of the steady-state kinetics data was carried out as previously described (29Silver 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 achieved by non-linear regression. When substrate inhibition was significant, data were analyzed using an extension of the Hill equation (30Pastra-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. Small-Angle X-ray Scattering—The SAXS experiments were performed on Beamline 4-2 at the Stanford Synchrotron Radiation Laboratory. The experimental set up and procedures were performed as described previously (31Sakash 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). Crystallization and Crystal Mounting—Crystals of the K244N mutant were obtained by the sitting drop method. Prior to crystallization the enzyme was dialyzed into 40 mm KH2PO4, 2.0 mm 2-mercaptoethanol, 0.2 mm EDTA, pH 7.0 for 24 h. Single crystals of the K244N mutant were obtained by mixing a 20 mg/ml filtered solution (0.22 μm) of the K244N enzyme with a solution of 17% (w/v) PEG 1450, 50 mm malonate, 0.2 mm EDTA, 1 mm sodium azide, and 20 mm Bis-Tris buffer, pH 7.0 in a 1:1 ratio (v/v). Rhombohedral-shaped crystals grew in ∼1 week. In preparation for data collection, crystals of dimensions ∼0.6 × 0.5 × 0.3 mm3 were sequentially transferred to a solution containing 20% PEG 1450, 20 mm Bis-Tris, pH 7.0, 50 mm malonate, 0.2 mm EDTA, 1 mm sodium azide, and 15% glycerol as cryoprotectant. Then the crystals were briefly dipped into a solution of 23% PEG 1450, 20 mm Bis-Tris, pH 7.0, 50 mm malonate, 0.2 mm EDTA, 1 mm sodium azide, and 20% glycerol as cryoprotectant. Crystals mounted in cryo-loops were frozen in liquid nitrogen and kept frozen until exposure to x-rays for data collection. Data Collection and Structure Refinement—All the crystallographic data used for structure determination in this work were collected on beamline X12C at 1 Å wavelength using the Brandeis-2k detector at 100 K at the National Synchrotron Light Source, Brookhaven National Laboratory. Data were collected up to 2.6 Å resolution, and analyzed using DENZO and SCALEPACK (32Otwinowski Z. Minor W. Carter Jr., C.W. Sweet R.M. Methods Enzymology. 276. Academic Press, NY1997: 307-326Google Scholar). The initial model used for refinement was the P268A mutant structure (PDB code: 1EZZ) (33Jin L. Stec B. Kantrowitz E.R. Biochemistry. 2000; 39: 8058-8066Crossref PubMed Scopus (24) Google Scholar). Although the crystals of the K244N mutant were of the same space group (R3) as the crystals of the P268A enzyme, there was a significant difference in the length of the a and b axes of the unit cell. After initial rigid body refinement and simulated annealing in CNS, it was apparent from an inflated Rfree value that the model bias was cumbersome. Therefore, a consensus model was built from overlaying several T state structures of aspartate transcarbamoylase (PDB codes 1EZZ, 1NBE, and 3AT1) using the program SEQUOIA (34Bruns C.M. Hubatsch I. Ridderstrom M. Mannervik B. Tainer J.A. J. Mol. Biol. 1999; 288: 427-439Crossref PubMed Scopus (156) Google Scholar) with all water molecules removed. Automated molecular replacement was performed using AmoRe in CCP4. This model was used for rigid body and simulated annealing refinement with initial bulk solvent correction with CNS (35Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges N. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16978) Google Scholar). Initial model rebuilding was performed using XtalView (36McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar). After several rounds of rebuilding and simulated annealing, the spread between the Rfactor and Rfree increased. Therefore, the TLS protocol utilizing tensor, libration and screw axis parameterization in CCP4 was employed. As previously described for intermediate resolution structures (37Schomaker V. Trueblood K.N. Acta Crystallogr. Sect. B. 1968; 24: 63-76Crossref Google Scholar, 38Schomaker V. Trueblood K.N. Acta Crystallogr. Sect. B,. 1998; 54: 507-514Crossref Scopus (134) Google Scholar), anisotropic displacement parameters were used to define a global parameterization of the asymmetric unit. This was done by defining the individual domains of the asymmetric unit as rigid bodies. This takes into account the tensor and libration parameters of the TLS refinement: both dynamic internal motions of the domains and static disorder, respectively. In addition, the screw axis parameter defined in the TLS matrices accounts for the screw axis of the dimer unit of the regulatory chains (of the asymmetric unit) not described by the crystallographic symmetry of R3. The non-crystallographic 2-fold axis of the regulatory dimer is also accounted for in the TLS matrices that were used. All of these factors were included in the calculation of the anisotropic displacement parameters and refinement statistics using REFMAC5 in CCP4. The TLS parameters were chosen to defined the domains of both the upper and lower catalytic and regulatory chains as rigid bodies. For the catalytic chains, residues 1:150 of the CP domain and residues 151:310 of the ASP domain with the C-terminal helix of the CP domain. For the regulatory chains, residues 1:100 of the AL domain and residues 101:153 of the ZN domain. Non-crystallographic symmetry describing the 2-fold axis of the regulatory dimers was also applied. After several rounds of refinement with REFMAC5 in CCP4, the refinement was continued with CNS. The positional refinement performed with the TLS protocol was preserved by defining rigid body groups as described above, using a simple energy minimization script and manual rebuilding with XtalView (36McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar). With the Rfactor and Rfree at 21 and 22%, respectively, and 98.8% of all side chains in allowed and generously allowed regions of the Ramachandran plot, solvent molecules were added manually. Water molecules were only added where they could be justified by hydrogen bonds and Fo-Fc electron density at or above the 2.5σ level. The final Rfactor and Rfree values were 20.1 and 23.1%, respectively. The data processing and refinement statistics are given in Table I.Table IData collection and refinement summary of the K244N structureData collectionSpace groupR3dmin (Å)2.6Total reflections230,763Unique reflections34,988Redundancy6.5Completeness (%) (all/outer shell)94.7/89.4Unit cell (Å)a = b = 125.667, c = 198.204Anglesα = β = 90°, γ = 120°Rmerge% (all/outer shell)a(∑hkl∑i|Imean−Ii|)/(∑hkl∑iIi)5.5/33.6RefinementResolution range (Å)29.0-2.6Sigma cutoff (σ)0Reflections30,529Average (I/σ)18.5Working R-factorBeginning0.274End0.201Free R-factorBeginning0.332End0.231Statistics (r.m.s. deviations)bStatistics were calculated in CNS with domains presented as rigid bodies to preserve the TLS assignmentsBonds (Å)0.010Angles (degrees)1.40Impropers (degrees)1.23Dihedrals (degrees)25.6a (∑hkl∑i|Imean−Ii|)/(∑hkl∑iIi)b Statistics were calculated in CNS with domains presented as rigid bodies to preserve the TLS assignments Open table in a new tab Steady-state Kinetics—The kinetic parameters calculated from the aspartate saturation curves (shown in Fig. 1) are displayed in Table II. Although the substitutions at position 244 have little influence on the maximal catalytic activity, both mutations cause dramatic reductions in cooperativity and the [Asp]0.5. The K244N enzyme exhibited a hyperbolic saturation curve with a maximal velocity of 18.7 mmol·h-1·mg-1, and an [Asp]0.5 of 4.0 mm. In contrast, the wild-type enzyme exhibited a sigmoidal aspartate saturation curve with a Hill coefficient of 2.5, a maximal velocity of 18.9 mmol·h-1·mg-1 and an [Asp]0.5 of 12.4 mm. The K244A enzyme displayed substantially reduced cooperativity, with a Hill coefficient of 1.2, a maximal velocity of 18.8 mmol·h-1·mg-1 and an [Asp]0.5 of 8.6 mm.Table IIKinetic parameters of the wild-type and mutant aspartate transcarbamoylases These data were determined from the aspartate saturation curves. Colorimetric assays were performed at 25 °C in 50 mm Tris acetate buffer, pH 8.3, and at a saturating concentration of carbamoyl phosphate (4.8 mm).EnzymeVmaxaMaximal observed specific activity[Asp]0.5nHmmol·h-1·mg-1mmWild type18.9 ± 1.4bErrors are the average deviation of three determinations12.4 ± 0.62.4 ± 0.2K244N18.7 ± 0.24.0 ± 0.21K244A18.8 ± 0.38.4 ± 0.21.2a Maximal observed specific activityb Errors are the average deviation of three determinations Open table in a new tab Influence of the Allosteric Effectors—The influence of the heterotropic effectors ATP and CTP were determined for the wild type as well as the K244N and K244A enzymes at one-half the [Asp]0.5. Nucleotides effects were determined at this aspartate concentration because the nucleotides exert a larger influence on the activity of the enzyme as the aspartate concentration is reduced (39Tauc P. Leconte C. Kerbiriou D. Thiry L. Hervé G. J. Mol. Biol. 1982; 155: 155-168Crossref PubMed Scopus (48) Google Scholar). From these nucleotide saturation curves, the maximal extent of activation or inhibition at infinite nucleotide concentration was calculated. The parameters were derived from the curves (shown in Fig. 2) and are displayed in Table III.Table IIIATP activation and CTP inhibition of the wild-type and mutant aspartate transcarbamoylases These data were determined from ATP and CTP saturation curves (Fig. 2). Colorimetric assays were performed at 25 °C in 50 mm Tris 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 enzyme at pH 8.3.EnzymeATPaPercent activation is defined as 100 (AATP/A) where AATP is the activity in the presence of ATP, and A is the activity in the absence of ATPKATPbK is the nucleotide concentration required to activate or inhibit the enzyme by 50% of the maximal effectCTPcPercent residual activity is defined as 100 (ACTP/A) where ACTP is the activity in the presence of CTP, and A is the activity in the absence of CTPKCTPbK is the nucleotide concentration required to activate or inhibit the enzyme by 50% of the maximal effect%mm%mmWild-type4000.25300.52K244N1200.63750.035K244A1400.61730.051a Percent activation is defined as 100 (AATP/A) where AATP is the activity in the presence of ATP, and A is the activity in the absence of ATPb K is the nucleotide concentration required to activate or inhibit the enzyme by 50% of the maximal effectc Percent residual activity is defined as 100 (ACTP/A) where ACTP is the activity in the presence of CTP, and A is the activity in the absence of CTP Open table in a new tab As shown in Fig. 2, the K244N and K244A enzymes exhibited similar activation by ATP and inhibition by CTP. However, the nucleotide effects for both mutant enzymes were much reduced as compared with the wild-type enzyme. The K244N and K244A enzymes were activated by ATP approximately one-fourth that of the wild-type enzyme, and were inhibited by CTP approximately one-third that of the wild-type enzyme. Effects of N-(phosphonoacetyl)-l-aspartate (PALA) on the K244N and K244A Enzymes—At low concentrations of aspartate, where the enzyme is predominately in the T state, low concentrations of PALA activate the wild-type enzyme (40Collins K.D. Stark G.R. J. Biol. Chem. 1971; 246: 6599-6605Abstract Full Text PDF PubMed Google Scholar) by shifting the population to the R state. At higher concentrations of PALA inhibition is observed as the remaining active sites are filled. For the K244N and K244A enzymes, there was no activation observed by PALA (data not shown). Small Angle X-ray Scattering—Small-angle x-ray scattering (SAXS) in solution was used to evaluate the quaternary structures of the wild-type, K244A and K244N enzymes in the absence and presence of saturating concentrations of carbamoyl phosphate and PALA at pH 8.3. For the wild-type enzyme, shown in Fig. 3A, there is a significant change in the SAXS pattern upon addition of PALA, with both a change in the peak position and an increase in relative intensity (41Moody M.F. Vachette P. Foote A.M. J. Mol. Biol. 1979; 133: 517-532Crossref PubMed Scopus (83) Google Scholar). A saturating concentration of carbamoyl phosphate (5 mm) only shifted the SAXS curve slightly as compared with the unliganded curve for the wild-type enzyme, indicating that CP binding induces only a small quaternary structural change. The SAXS scattering curves for the unliganded K244A and K244N enzymes, shown in Fig. 3, B and C, respectively, are shifted slightly toward the R state as compared with the T state SAXS pattern of the wild-type enzyme. In the presence of PALA, the SAXS curves of both the K244A and K244N enzymes are nearly identical to the SAXS curve of the wild-type R state. Unlike the wild-type enzyme, the addition of a saturating concentration of carbamoyl phosphate (5 mm) significantly shifted the SAXS curves for both the K244A and K244N enzymes. For the K244A enzyme, the addition of CP shifted the structure nearly all the way to the R state. However, for the K244N enzyme the addition of CP shifted the structure all the way to the R state. Three-dimensional Structure of the K244N Enzyme—The K244N enzyme in the absence of ligands was crystallized in the R3 space group with unit cell dimensions of a = b = 125.67, c = 198.2 (Table I). The K244N enzyme had similar unit cell dimensions along the c axis and the same space group as observed for crystals of the P268A aspartate transcarbamoylase that was crystallized under similar conditions (33Jin L. Stec B. Kantrowitz E.R. Biochemistry. 2000; 39: 8058-8066Crossref PubMed Scopus (24) Google Scholar). However, the length of the a and b axes were ∼4 Å shorter than that observed for the crystal of the P268A enzyme. The crystals of the K244N enzyme diffracted to 2.6 Å resolution with completeness of ∼95% (Table I), and the structure was refined to an Rfactor and Rfree of 20.1 and 23.1%, respectively, with 50 waters. The vertical separation of the two catalytic trimers of aspartate transcarbamoylase is defined as the distance between the center of mass of the C1 2Within the E. coli holoenzyme, the catalytic chains of the top catalytic trimer are numbered C1, C2, and C3, whereas the catalytic chains of the bottom catalytic trimer are numbered C4, C5, and C6, with C4 under C1. The regulatory dimers contain chains R1-R6, R2-R4, and R3-R5. A regulatory chain is in direct co" @default.
• W1968313869 created "2016-06-24" @default.
• W1968313869 creator A5052240170 @default.
• W1968313869 creator A5059952544 @default.
• W1968313869 creator A5062676789 @default.
• W1968313869 creator A5065689974 @default.
• W1968313869 creator A5087356818 @default.
• W1968313869 creator A5090422616 @default.
• W1968313869 date "2004-05-01" @default.
• W1968313869 modified "2023-10-17" @default.
• W1968313869 title "240s Loop Interactions Stabilize the T State of Escherichia coli Aspartate Transcarbamoylase" @default.
• W1968313869 cites W1496116773 @default.
• W1968313869 cites W1506226772 @default.
• W1968313869 cites W1545540883 @default.
• W1968313869 cites W1555002836 @default.
• W1968313869 cites W1561167748 @default.
• W1968313869 cites W1568502048 @default.
• W1968313869 cites W1606830445 @default.
• W1968313869 cites W1964370928 @default.
• W1968313869 cites W1969542803 @default.
• W1968313869 cites W1972395614 @default.
• W1968313869 cites W1977874193 @default.
• W1968313869 cites W1978181627 @default.
• W1968313869 cites W1979013212 @default.
• W1968313869 cites W1979332372 @default.
• W1968313869 cites W1979503548 @default.
• W1968313869 cites W1981301590 @default.
• W1968313869 cites W1985937560 @default.
• W1968313869 cites W1990650142 @default.
• W1968313869 cites W1995017064 @default.
• W1968313869 cites W1997972388 @default.
• W1968313869 cites W2005392755 @default.
• W1968313869 cites W2007031737 @default.
• W1968313869 cites W2010649113 @default.
• W1968313869 cites W2015999520 @default.
• W1968313869 cites W2016121726 @default.
• W1968313869 cites W2018225090 @default.
• W1968313869 cites W2032494141 @default.
• W1968313869 cites W2033850649 @default.
• W1968313869 cites W2035505895 @default.
• W1968313869 cites W2039369222 @default.
• W1968313869 cites W2047497909 @default.
• W1968313869 cites W2047595408 @default.
• W1968313869 cites W2069204236 @default.
• W1968313869 cites W2073609299 @default.
• W1968313869 cites W2076325559 @default.
• W1968313869 cites W2080736184 @default.
• W1968313869 cites W2081076206 @default.
• W1968313869 cites W2088594784 @default.
• W1968313869 cites W2093945608 @default.
• W1968313869 cites W2095053750 @default.
• W1968313869 cites W2097758310 @default.
• W1968313869 cites W2100837269 @default.
• W1968313869 cites W2104091052 @default.
• W1968313869 cites W2116026514 @default.
• W1968313869 cites W2125630053 @default.
• W1968313869 cites W2132068224 @default.
• W1968313869 cites W2132832161 @default.
• W1968313869 cites W2135839939 @default.
• W1968313869 cites W2152255924 @default.
• W1968313869 cites W2159716299 @default.
• W1968313869 cites W2322719150 @default.
• W1968313869 cites W4293247451 @default.
• W1968313869 doi "https://doi.org/10.1074/jbc.m401637200" @default.
• W1968313869 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15014067" @default.
• W1968313869 hasPublicationYear "2004" @default.
• W1968313869 type Work @default.
• W1968313869 sameAs 1968313869 @default.
• W1968313869 citedByCount "4" @default.
• W1968313869 countsByYear W19683138692015 @default.
• W1968313869 crossrefType "journal-article" @default.
• W1968313869 hasAuthorship W1968313869A5052240170 @default.
• W1968313869 hasAuthorship W1968313869A5059952544 @default.
• W1968313869 hasAuthorship W1968313869A5062676789 @default.
• W1968313869 hasAuthorship W1968313869A5065689974 @default.
• W1968313869 hasAuthorship W1968313869A5087356818 @default.
• W1968313869 hasAuthorship W1968313869A5090422616 @default.
• W1968313869 hasBestOaLocation W19683138691 @default.
• W1968313869 hasConcept C104317684 @default.
• W1968313869 hasConcept C114614502 @default.
• W1968313869 hasConcept C121332964 @default.
• W1968313869 hasConcept C184670325 @default.
• W1968313869 hasConcept C185592680 @default.
• W1968313869 hasConcept C197248121 @default.
• W1968313869 hasConcept C2908821069 @default.
• W1968313869 hasConcept C33923547 @default.
• W1968313869 hasConcept C547475151 @default.
• W1968313869 hasConcept C55493867 @default.
• W1968313869 hasConcept C8010536 @default.
• W1968313869 hasConceptScore W1968313869C104317684 @default.
• W1968313869 hasConceptScore W1968313869C114614502 @default.
• W1968313869 hasConceptScore W1968313869C121332964 @default.
• W1968313869 hasConceptScore W1968313869C184670325 @default.
• W1968313869 hasConceptScore W1968313869C185592680 @default.
• W1968313869 hasConceptScore W1968313869C197248121 @default.
• W1968313869 hasConceptScore W1968313869C2908821069 @default.
• W1968313869 hasConceptScore W1968313869C33923547 @default.
• W1968313869 hasConceptScore W1968313869C547475151 @default.

• next