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Carbamoyl Phosphate (carbamoyl + phosphate)
Selected AbstractsLong-range allosteric transitions in carbamoyl phosphate synthetasePROTEIN SCIENCE, Issue 9 2004James B. Thoden Abstract Carbamoyl phosphate synthetase plays a key role in both pyrimidine and arginine biosynthesis by catalyzing the production of carbamoyl phosphate from one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli consists of two polypeptide chains referred to as the small and large subunits, which contain a total of three separate active sites that are connected by an intramolecular tunnel. The small subunit harbors one of these active sites and is responsible for the hydrolysis of glutamine to glutamate and ammonia. The large subunit binds the two required molecules of MgATP and is involved in assembling the final product. Compounds such as L-ornithine, UMP, and IMP allosterically regulate the enzyme. Here, we report the three-dimensional structure of a site-directed mutant protein of carbamoyl phosphate synthetase from E. coli, where Cys 248 in the small subunit was changed to an aspartate. This residue was targeted for a structural investigation because previous studies demonstrated that the partial glutaminase activity of the C248D mutant protein was increased 40-fold relative to the wild-type enzyme, whereas the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished. Remarkably, although Cys 248 in the small subunit is located at ,100 Å from the allosteric binding pocket in the large subunit, the electron density map clearly revealed the presence of UMP, although this ligand was never included in the purification or crystallization schemes. The manner in which UMP binds to carbamoyl phosphate synthetase is described. [source] Refined structure of Pyrococcus furiosus ornithine carbamoyltransferase at 1.87,AACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003Jan Massant Using synchrotron radiation, X-ray data have been collected from Pyrococcus furiosus ornithine carbamoyltransferase (Pfu OTCase) to a maximal resolution of 1.87,Å, allowing the refinement of a previous structure at 2.7,Å [Villeret et al. (1998), Proc. Natl Acad. Sci. USA, 95, 2801,2806]. Thanks to the high resolution of this refined structure, two sulfate ions and 191 water molecules could be localized directly from the electron-density maps. The identification of these molecules allowed a more rigorous description of the active site and the identification of residues involved in binding carbamoyl phosphate. The improved quality of the model resulted in a better definition of several loops and the various interfaces. The dodecameric protein is composed of four catalytic trimers disposed in a tetrahedral manner. The extreme thermal stability of Pfu OTCase is mainly the result of the strengthening of the intersubunit interactions in a trimer and oligomerization of the trimers into a dodecamer. Interfaces between monomers in a catalytic trimer are characterized by an increase in ion-pair networks compared with mesophilic OTCases. However, the interfaces between catalytic trimers in the dodecameric oligomer are mainly hydrophobic and also involve aromatic,aromatic and cation,, interactions. [source] Crystallization and structure determination of the catalytic trimer of Methanococcus jannaschii aspartate transcarbamoylaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2000Jacqueline Vitali Aspartate transcarbamoylase (ATCase) catalyzes the first step in the pyrimidine biosynthetic pathway, the reaction between carbamoyl phosphate and l -aspartate to form N -carbamoyl- l -aspartate and phosphate. The structural analysis of the ATCase catalytic trimer from Methanococcus jannaschii, a unicellular thermophilic archaeabacterium, has been undertaken in order to gain insight into the structural features that are responsible for the thermostability of the enzyme. As a first step, the catalytic trimer was crystallized in space group R32, with unit-cell parameters a = b = 265.3, c = 195.5,Å and two trimers in the asymmetric unit. Its structure was determined using molecular replacement and Patterson methods. In general, structures containing multiple copies of molecules in the asymmetric unit are difficult to determine. In this case, the two trimers in the asymmetric unit are parallel to each other and use of the Patterson function greatly simplified the structure solution. [source] X-ray structure and characterization of carbamate kinase from the human parasite Giardia lambliaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Andrey Galkin Carbamate kinase catalyzes the reversible conversion of carbamoyl phosphate and ADP to ATP and ammonium carbamate, which is hydrolyzed to ammonia and carbonate. The three-dimensional structure of carbamate kinase from the human parasite Giardia lamblia (glCK) has been determined at 3,Å resolution. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 69.77, b = 85.41, c = 102.1,Å, , = 106.8°. The structure was refined to a final R factor of 0.227. The essentiality of glCK together with its absence in humans makes the enzyme an attractive candidate for anti- Giardia drug development. Steady-state kinetic rate constants have been determined. The kcat for ATP formation is 319 ± 9,s,1. The Km values for carbamoyl phosphate and ADP are 85 ± 6 and 70 ± 5,µM, respectively. The structure suggests that three invariant lysine residues (Lys131, Lys216 and Lys278) may be involved in the binding of substrates and phosphoryl transfer. The structure of glCK reveals that a glycerol molecule binds in the likely carbamoyl phosphate-binding site. [source] Crystallization and preliminary X-ray crystallographic analysis of the [NiFe]-hydrogenase maturation factor HypF1 from Ralstonia eutropha H16ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Gordon Winter The hydrogenase maturation factor HypF1 is a truncated but functional version of the HypF protein. HypF is known to be involved in the supply of the CN, ligands of the active site of [NiFe]-hydrogenases, utilizing carbamoyl phosphate as a substrate. The first crystallization and preliminary X-ray studies of HypF1 from Ralstonia eutropha H16 are reported here. Crystals of HypF1 (394 amino acids, 40.7,kDa) were obtained by the sitting-drop vapour-diffusion technique using sodium formate as a precipitant. The crystals belonged to space group I222, with unit-cell parameters a = 79.7, b = 91.6, c = 107.2,Å. Complete X-ray diffraction data sets were collected at 100,K from native crystals and from a platinum derivative to a maximum resolution of 1.65,Å. [source] Crystallization and preliminary X-ray analysis of aspartate transcarbamoylase from the parasitic protist Trypanosoma cruziACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Kazuaki Matoba Aspartate transcarbamoylase (ATCase), the second enzyme of the de novo pyrimidine-biosynthetic pathway, catalyzes the production of carbamoyl aspartate from carbamoyl phosphate and l -aspartate. In contrast to Escherichia coli ATCase and eukaryotic CAD multifunctional fusion enzymes, Trypanosoma cruzi ATCase lacks regulatory subunits and is not part of the multifunctional fusion enzyme. Recombinant T. cruzi ATCase expressed in E. coli was purified and crystallized in a ligand-free form and in a complex with carbamoyl phosphate at 277,K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. Ligand-free crystals (space group P1, unit-cell parameters a = 78.42, b = 79.28, c = 92.02,Å, , = 69.56, , = 82.90, , = 63.25°) diffracted X-rays to 2.8,Å resolution, while those cocrystallized with carbamoyl phosphate (space group P21, unit-cell parameters a = 88.41, b = 158.38, c = 89.00,Å, , = 119.66°) diffracted to 1.6,Å resolution. The presence of two homotrimers in the asymmetric unit (38,kDa × 6) gives VM values of 2.3 and 2.5,Å3,Da,1 for the P1 and P21 crystal forms, respectively. [source] | ||||||||||