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Guy Hervé - One of the best experts on this subject based on the ideXlab platform.

  • pressure induced dissociation of Carbamoyl phosphate synthetase domains the catalytically active form is dimeric
    Journal of Biological Chemistry, 1998
    Co-Authors: Hedeel I. Guy, Guy Hervé, Bernard Schmidt, David R. Evans
    Abstract:

    Carbamoyl-phosphate synthetase consists of an amidotransferase domain or subunit (GLN) that hydrolyzes glutamine and transfers the ammonia to the synthetase component (CPS) where the biosynthetic reaction occurs. The CPS domain is composed of two homologous subdomains, CPS.A and CPS.B, that catalyze different ATP-dependent reactions involved in Carbamoyl phosphate synthesis. When the individual CPS.A and CPS.B subdomains were individually cloned and expressed in Escherichia coli (Guy, H. I., and Evans, D. R. (1996) J. Biol. Chem. 271, 13762-13769), they were found to be functionally equivalent and could each independently catalyze Carbamoyl phosphate synthesis. The proposal was advanced that, although the monomers could catalyze the individual partial reactions, overall synthesis of Carbamoyl phosphate required a homodimer of CPS.A or CPS.B. To test this hypothesis, the GLN-CPS.B dimer was reversibly dissociated at 1500 bar in a high pressure cell. Dissociation was accompanied by a loss of both glutamine- and ammonia-dependent CPSase activity. Activity was recovered once the protein was returned to atmospheric pressure. If the sample was cross-linked before exposure to high pressure, there was no dissociation and no loss of biosynthetic activity. In contrast, the bicarbonate-dependent ATPase and the Carbamoyl phosphate-dependent ATP synthetase activities were largely unaffected by pressure-induced dissociation. These experiments confirmed the hypothesis that the synthesis of Carbamoyl phosphate requires the concerted action of the two active sites within the homodimer.

  • Purification and Characterization of Carbamoyl‐Phosphate Synthetase from the Deep‐Sea Hyperthermophilic Archaebacterium Pyrococcus abyssi
    FEBS Journal, 1996
    Co-Authors: Cristina Purcarea, Daniel Prieur, Valérie Simon, Guy Hervé
    Abstract:

    Carbamoyl-phosphate synthetase was purified from the deep-sea hyperthermophilic archaebacterium Pyrococcus abyssi. This enzyme appears to be monomeric and uses ammonium salts as nitrogen donor. Its activity is inhibited by some nucleotides that compete with ATP. In contrast with the Carbamoyl-phosphate synthetases investigated so far, this enzyme is very resistant to high temperature. Its low molecular mass (46.6 kDa) and its catalytic properties suggest that the gene coding for this enzyme is a previously postulated ancestor whose duplication gave the genes coding for Carbamoyl-phosphate synthetases and carbamate kinases.

  • purification and characterization of Carbamoyl phosphate synthetase from the deep sea hyperthermophilic archaebacterium pyrococcus abyssi
    FEBS Journal, 1996
    Co-Authors: Cristina Purcarea, Daniel Prieur, Valérie Simon, Guy Hervé
    Abstract:

    Carbamoyl-phosphate synthetase was purified from the deep-sea hyperthermophilic archaebacterium Pyrococcus abyssi. This enzyme appears to be monomeric and uses ammonium salts as nitrogen donor. Its activity is inhibited by some nucleotides that compete with ATP. In contrast with the Carbamoyl-phosphate synthetases investigated so far, this enzyme is very resistant to high temperature. Its low molecular mass (46.6 kDa) and its catalytic properties suggest that the gene coding for this enzyme is a previously postulated ancestor whose duplication gave the genes coding for Carbamoyl-phosphate synthetases and carbamate kinases.

Cristina Purcarea - One of the best experts on this subject based on the ideXlab platform.

Hazel M. Holden - One of the best experts on this subject based on the ideXlab platform.

  • The structure of Carbamoyl phosphate synthetase determined to 2.1 Å resolution
    Acta Crystallographica Section D Biological Crystallography, 1999
    Co-Authors: James B. Thoden, Frank M. Raushel, Matthew M. Benning, Ivan Rayment, Hazel M. Holden
    Abstract:

    Carbamoyl phosphate synthetase catalyzes the formation of Carbamoyl phosphate from one molecule of bicarbonate, two molecules of Mg2+ATP and one molecule of glutamine or ammonia depending upon the particular form of the enzyme under investigation. As isolated from Escherichia coli, the enzyme is an \alpha,β-heterodimer consisting of a small subunit that hydrolyzes glutamine and a large subunit that catalyzes the two required phosphorylation events. Here the three-dimensional structure of Carbamoyl phosphate synthetase from E. coli refined to 2.1 A resolution with an R factor of 17.9% is described. The small subunit is distinctly bilobal with a catalytic triad (Cys269, His353 and Glu355) situated between the two structural domains. As observed in those enzymes belonging to the \alpha/\beta-hydrolase family, the active-site nucleophile, Cys269, is perched at the top of a tight turn. The large subunit consists of four structural units: the carboxyphosphate synthetic component, the oligomerization domain, the Carbamoyl phosphate synthetic component and the allosteric domain. Both the carboxyphosphate and Carbamoyl phosphate synthetic components bind Mn2+ADP. In the carboxyphosphate synthetic component, the two observed Mn2+ ions are both octahedrally coordinated by oxygen-containing ligands and are bridged by the carboxylate side chain of Glu299. Glu215 plays a key allosteric role by coordinating to the physiologically important potassium ion and hydrogen bonding to the ribose hydroxyl groups of ADP. In the Carbamoyl phosphate synthetic component, the single observed Mn2+ ion is also octahedrally coordinated by oxygen-containing ligands and Glu761 plays a similar role to that of Glu215. The carboxy­phosphate and Carbamoyl phosphate synthetic components, while topologically equivalent, are structurally different, as would be expected in light of their separate biochemical functions.

  • The structure of Carbamoyl phosphate synthetase determined to 2.1 A resolution.
    Acta crystallographica. Section D Biological crystallography, 1999
    Co-Authors: James B. Thoden, Frank M. Raushel, Matthew M. Benning, Ivan Rayment, Hazel M. Holden
    Abstract:

    Carbamoyl phosphate synthetase catalyzes the formation of Carbamoyl phosphate from one molecule of bicarbonate, two molecules of Mg2+ATP and one molecule of glutamine or ammonia depending upon the particular form of the enzyme under investigation. As isolated from Escherichia coli, the enzyme is an alpha,beta-heterodimer consisting of a small subunit that hydrolyzes glutamine and a large subunit that catalyzes the two required phosphorylation events. Here the three-dimensional structure of Carbamoyl phosphate synthetase from E. coli refined to 2.1 A resolution with an R factor of 17.9% is described. The small subunit is distinctly bilobal with a catalytic triad (Cys269, His353 and Glu355) situated between the two structural domains. As observed in those enzymes belonging to the alpha/beta-hydrolase family, the active-site nucleophile, Cys269, is perched at the top of a tight turn. The large subunit consists of four structural units: the carboxyphosphate synthetic component, the oligomerization domain, the Carbamoyl phosphate synthetic component and the allosteric domain. Both the carboxyphosphate and Carbamoyl phosphate synthetic components bind Mn2+ADP. In the carboxyphosphate synthetic component, the two observed Mn2+ ions are both octahedrally coordinated by oxygen-containing ligands and are bridged by the carboxylate side chain of Glu299. Glu215 plays a key allosteric role by coordinating to the physiologically important potassium ion and hydrogen bonding to the ribose hydroxyl groups of ADP. In the Carbamoyl phosphate synthetic component, the single observed Mn2+ ion is also octahedrally coordinated by oxygen-containing ligands and Glu761 plays a similar role to that of Glu215. The carboxyphosphate and Carbamoyl phosphate synthetic components, while topologically equivalent, are structurally different, as would be expected in light of their separate biochemical functions.

David R. Evans - One of the best experts on this subject based on the ideXlab platform.

  • Functional linkage between the glutaminase and synthetase domains of Carbamoyl-phosphate synthetase. Role of serine 44 in Carbamoyl-phosphate synthetase-aspartate Carbamoyltransferase-dihydroorotase (cad).
    The Journal of biological chemistry, 1999
    Co-Authors: Anura Hewagama, Hedeel I. Guy, John F. Vickrey, David R. Evans
    Abstract:

    Mammalian Carbamoyl-phosphate synthetase is part of Carbamoyl-phosphate synthetase-aspartate Carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that also catalyzes the second and third steps of pyrimidine biosynthesis. Carbamoyl phosphate synthesis requires the concerted action of the glutaminase (GLN) and Carbamoyl-phosphate synthetase domains of CAD. There is a functional linkage between these domains such that glutamine hydrolysis on the GLN domain does not occur at a significant rate unless ATP and HCO3−, the other substrates needed for Carbamoyl phosphate synthesis, bind to the synthetase domain. The GLN domain consists of catalytic and attenuation subdomains. In the separately cloned GLN domain, the catalytic subdomain is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage. Replacement of Ser44 in the GLN attenuation domain with alanine increases thekcat/Km for glutamine hydrolysis 680-fold. The formation of a functional hybrid between the mammalian Ser44 GLN domain and the Escherichia coli Carbamoyl-phosphate synthetase large subunit had little effect on glutamine hydrolysis. In contrast, ATP and HCO3− did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted. In accord with this interpretation, the rate of glutamine hydrolysis and Carbamoyl phosphate synthesis were no longer coordinated. Approximately 3 times more glutamine was hydrolyzed by the Ser44 → Ala mutant than that needed for Carbamoyl phosphate synthesis. Ser44, the only attenuation subdomain residue that extends into the GLN active site, appears to be an integral component of the regulatory circuit that phases glutamine hydrolysis and Carbamoyl phosphate synthesis.

  • pressure induced dissociation of Carbamoyl phosphate synthetase domains the catalytically active form is dimeric
    Journal of Biological Chemistry, 1998
    Co-Authors: Hedeel I. Guy, Guy Hervé, Bernard Schmidt, David R. Evans
    Abstract:

    Carbamoyl-phosphate synthetase consists of an amidotransferase domain or subunit (GLN) that hydrolyzes glutamine and transfers the ammonia to the synthetase component (CPS) where the biosynthetic reaction occurs. The CPS domain is composed of two homologous subdomains, CPS.A and CPS.B, that catalyze different ATP-dependent reactions involved in Carbamoyl phosphate synthesis. When the individual CPS.A and CPS.B subdomains were individually cloned and expressed in Escherichia coli (Guy, H. I., and Evans, D. R. (1996) J. Biol. Chem. 271, 13762-13769), they were found to be functionally equivalent and could each independently catalyze Carbamoyl phosphate synthesis. The proposal was advanced that, although the monomers could catalyze the individual partial reactions, overall synthesis of Carbamoyl phosphate required a homodimer of CPS.A or CPS.B. To test this hypothesis, the GLN-CPS.B dimer was reversibly dissociated at 1500 bar in a high pressure cell. Dissociation was accompanied by a loss of both glutamine- and ammonia-dependent CPSase activity. Activity was recovered once the protein was returned to atmospheric pressure. If the sample was cross-linked before exposure to high pressure, there was no dissociation and no loss of biosynthetic activity. In contrast, the bicarbonate-dependent ATPase and the Carbamoyl phosphate-dependent ATP synthetase activities were largely unaffected by pressure-induced dissociation. These experiments confirmed the hypothesis that the synthesis of Carbamoyl phosphate requires the concerted action of the two active sites within the homodimer.

Valérie Simon - One of the best experts on this subject based on the ideXlab platform.