Publications

• The amidotransferase family of enzymes: molecular machines for the production and delivery of ammonia.

  Raushel F.M., Thoden J.B., Holden H.M.

  The amidotransferase family of enzymes utilizes the ammonia derived from the hydrolysis of glutamine for a subsequent chemical reaction catalyzed by the same enzyme. The ammonia intermediate does not dissociate into solution during the chemical transformations. A well-characterized example of the ... >> More

  The amidotransferase family of enzymes utilizes the ammonia derived from the hydrolysis of glutamine for a subsequent chemical reaction catalyzed by the same enzyme. The ammonia intermediate does not dissociate into solution during the chemical transformations. A well-characterized example of the structure and mechanism displayed by this class of enzymes is provided by carbamoyl phosphate synthetase (CPS). Carbamoyl phosphate synthetase is isolated from Escherichia coli as a heterodimeric protein. The smaller of the two subunits catalyzes the hydrolysis of glutamine to glutamate and ammonia. The larger subunit catalyzes the formation of carbamoyl phosphate using 2 mol of ATP, bicarbonate, and ammonia. Kinetic investigations have led to a proposed chemical mechanism for this enzyme that requires carboxy phosphate, ammonia, and carbamate as kinetically competent reaction intermediates. The three-dimensional X-ray crystal structure of CPS has localized the positions of three active sites. The nucleotide binding site within the N-terminal half of the large subunit is required for the phosphorylation of bicarbonate and subsequent formation of carbamate. The nucleotide binding site within the C-terminal domain of the large subunit catalyzes the phosphorylation of carbamate to the final product, carbamoyl phosphate. The three active sites within the heterodimeric protein are separated from one another by about 45 A. The ammonia produced within the active site of the small subunit is the substrate for reaction with the carboxy phosphate intermediate that is formed in the active site found within the N-terminal half of the large subunit of CPS. Since the ammonia does not dissociate from the protein prior to its reaction with carboxy phosphate, this intermediate must therefore diffuse through a molecular tunnel that connects these two sites with one another. Similarly, the carbamate intermediate, initially formed at the active site within the N-terminal half of the large subunit, is the substrate for phosphorylation by the ATP bound to the active site located in the C-terminal half of the large subunit. A molecular passageway has been identified by crystallographic methods that apparently facilitates diffusion between these two active sites within the large subunit of CPS. Synchronization of the chemical transformations is controlled by structural perturbations among the three active sites. Molecular tunnels between distant active sites have also been identified in tryptophan synthase and glutamine phosphoribosyl pyrophosphate amidotransferase and are likely architectural features in an expanding list of enzymes. << Less

  Biochemistry 38:7891-7899(1999) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Purification of carbamyl phosphate synthetase from frog liver.

  MARSHALL M., METZENBERG R.L., COHEN P.P.

  J Biol Chem 233:102-105(1958) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Carbamoyl phosphate synthetase: a tunnel runs through it.

  Holden H.M., Thoden J.B., Raushel F.M.

  The direct transfer of metabolites from one protein to another in a biochemical pathway or between one active site and another within a single enzyme has been described as substrate channeling. The first structural visualization of such a phenomenon was provided by the X-ray crystallographic analy ... >> More

  The direct transfer of metabolites from one protein to another in a biochemical pathway or between one active site and another within a single enzyme has been described as substrate channeling. The first structural visualization of such a phenomenon was provided by the X-ray crystallographic analysis of tryptophan synthase, in which a tunnel of approximately 25 A in length was observed. The recently determined three-dimensional structure of carbamoyl phosphate synthetase sets a new long distance record in that the three active sites are separated by nearly 100 A. << Less

  Curr Opin Struct Biol 8:679-685(1998) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Carbamoyl phosphate synthetase: a crooked path from substrates to products.

  Raushel F.M., Thoden J.B., Reinhart G.D., Holden H.M.

  The formation of carbamoyl phosphate is catalyzed by a single enzyme using glutamine, bicarbonate and two molecules of ATP via a reaction mechanism that requires a minimum of four consecutive reactions and three unstable intermediates. The recently determined X-ray crystal structure of carbamoyl p ... >> More

  The formation of carbamoyl phosphate is catalyzed by a single enzyme using glutamine, bicarbonate and two molecules of ATP via a reaction mechanism that requires a minimum of four consecutive reactions and three unstable intermediates. The recently determined X-ray crystal structure of carbamoyl phosphate synthetase has revealed the location of three separate active sites connected by two molecular tunnels that run through the interior of the protein. It has been demonstrated that the amidotransferase domain within the small subunit of the enzyme from Escherichia coli hydrolyzes glutamine to ammonia via a thioester intermediate with Cys269. The ammonia migrates through the interior of the protein, where it reacts with carboxy phosphate to produce the carbamate intermediate. The carboxy phosphate intermediate is formed by the phosphorylation of bicarbonate by ATP at a site contained within the amino-terminal half of the large subunit. The carbamate intermediate is transported through the interior of the protein to a second site within the carboxy-terminal half of the large subunit, where it is phosphorylated by another ATP to yield the final product, carbamoyl phosphate. The entire journey from substrate to product covers a distance of nearly 100 A. << Less

  Curr Opin Chem Biol 2:624-632(1998) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Structural insight on the control of urea synthesis: identification of the binding site for N-acetyl-L-glutamate, the essential allosteric activator of mitochondrial carbamoyl phosphate synthetase.

  Pekkala S., Martinez A.I., Barcelona B., Gallego J., Bendala E., Yefimenko I., Rubio V., Cervera J.

  NAG (N-acetyl-L-glutamate), the essential allosteric activator of the first urea cycle enzyme, CPSI (carbamoyl phosphate synthetase I), is a key regulator of this crucial cycle for ammonia detoxification in animals (including humans). Automated cavity searching and flexible docking have allowed id ... >> More

  NAG (N-acetyl-L-glutamate), the essential allosteric activator of the first urea cycle enzyme, CPSI (carbamoyl phosphate synthetase I), is a key regulator of this crucial cycle for ammonia detoxification in animals (including humans). Automated cavity searching and flexible docking have allowed identification of the NAG site in the crystal structure of human CPSI C-terminal domain. The site, a pocket lined by invariant residues and located between the central beta-sheet and two alpha-helices, opens at the beta-sheet C-edge and is roofed by a three-residue lid. It can tightly accommodate one extended NAG molecule having the delta-COO- at the pocket entry, the alpha-COO- and acetamido groups tightly hydrogen bonded to the pocket, and the terminal methyl of the acetamido substituent surrounded by hydrophobic residues. This binding mode is supported by the observation of reduced NAG affinity upon mutation of NAG-interacting residues of CPSI (recombinantly expressed using baculovirus/insect cells); by the fine-mapping of the N-chloroacetyl-L-glutamate photoaffinity labelling site of CPSI; and by previously established structure-activity relationships for NAG analogues. The location of the NAG site is identical to that of the weak bacterial CPS activator IMP (inosine monophosphate) in Escherichia coli CPS, indicating a common origin for these sites and excluding any relatedness to the binding site of the other bacterial CPS activator, ornithine. Our findings open the way to the identification of CPSI deficiency patients carrying NAG site mutations, and to the possibility of tailoring the activator to fit a given NAG site mutation, as exemplified here with N-acetyl-L(+/-)-beta-phenylglutamate for the W1410K CPSI mutation. << Less

  Biochem. J. 424:211-220(2009) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The pathway of carbonate in the biosynthesis of carbamyl phosphate.

  JONES M.E., SPECTOR L.

  J Biol Chem 235:2897-2901(1960) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Carbamoyl-phosphate synthetase. Creation of an escape route for ammonia.

  Thoden J.B., Huang X., Raushel F.M., Holden H.M.

  Carbamoyl-phosphate synthetase catalyzes the production of carbamoyl phosphate through a reaction mechanism requiring one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli is composed of two polypeptide chains. The smaller of these bel ... >> More

  Carbamoyl-phosphate synthetase catalyzes the production of carbamoyl phosphate through a reaction mechanism requiring one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli is composed of two polypeptide chains. The smaller of these belongs to the Class I amidotransferase superfamily and contains all of the necessary amino acid side chains required for the hydrolysis of glutamine to glutamate and ammonia. Two homologous domains from the larger subunit adopt conformations that are characteristic for members of the ATP-grasp superfamily. Each of these ATP-grasp domains contains an active site responsible for binding one molecule of MgATP. High resolution x-ray crystallographic analyses have shown that, remarkably, the three active sites in the E. coli enzyme are connected by a molecular tunnel of approximately 100 A in total length. Here we describe the high resolution x-ray crystallographic structure of the G359F (small subunit) mutant protein of carbamoyl phosphate synthetase. This residue was initially targeted for study because it resides within the interior wall of the molecular tunnel leading from the active site of the small subunit to the first active site of the large subunit. It was anticipated that a mutation to the larger residue would "clog" the ammonia tunnel and impede the delivery of ammonia from its site of production to the site of utilization. In fact, the G359F substitution resulted in a complete change in the conformation of the loop delineated by Glu-355 to Ala-364, thereby providing an "escape" route for the ammonia intermediate directly to the bulk solvent. The substitution also effected the disposition of several key catalytic amino acid side chains in the small subunit active site. << Less

  J Biol Chem 277:39722-39727(2002) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Physical and Kinetic Properties of Carbamyl Phosphate Synthetase from Frog Liver.

  Marshall M., Metzenberg R.L., Cohen P.P.

  J Biol Chem 236:2229-2237(1961) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• A KINETIC STUDY OF CARBAMYL PHOSPHATE SYNTHETASE.

  FAHIEN L.A., COHEN P.P.

  J Biol Chem 239:1925-1934(1964) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Human carbamylphosphate synthetase I. Stabilization, purification, and partial characterization of the enzyme from human liver.

  Pierson D.L., Brien J.M.

  Carbamylphosphate synthetase I from human liver was stabilized, purified, and partially characterized. The labile enzyme was stabilized in cell-free extracts by the presence of MgATP and dithiothreitol at pH 7.8. The stabilized enzyme was purified by a rapid procedure consisting of ion exchange ch ... >> More

  Carbamylphosphate synthetase I from human liver was stabilized, purified, and partially characterized. The labile enzyme was stabilized in cell-free extracts by the presence of MgATP and dithiothreitol at pH 7.8. The stabilized enzyme was purified by a rapid procedure consisting of ion exchange chromatograhy and electrofocusing The native molecular weight of the enzyme was determined by gel filtration to be 190,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a monomeric molecular weight of 165,000. The isoelectric point of the purified enzyme was 6.05, and only one species of active enzyme was observed during electrofocusing of both purified enzyme preparations and crude liver homogenates. The enzyme exhibited a pH optimum of 7.8. The apparent Michaelis constants for NH4+, HCO3-, MgATP, and the activator, N-acetyl-L-glutamic acid, were 0.8, 6.7, 1.1, and 0.1 mM, respectively. << Less

  J Biol Chem 255:7891-7895(1980) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.