Catalytically active monomer and dimer forms of rat liver carbamoyl-phosphate synthetase.
Purified carbamoyl-phosphate synthetase of rat liver is shown to exist in a state of rapid, reversible monomer-dimer equilibrium. The allosteric activator N-acetyl-L-glutamate displaces the equilibrium toward monomer formation. This effect is observed over a range of initial protein concentrations of 0.02-5 mg/mL. Measurements of Stokes radii by analytical gel chromatography indicate that at concentrations less than 0.1 mg/mL at 25 degrees C in the presence of all the substrates the enzyme exists as a monomer of 160000 molecular weight. A gel chromatographic method was developed to identify the active form of carbamoyl-phosphate synthetase. On the basis of analysis of the ADP boundary formed during gel chromatography, the monomer is established to be catalytically active. Active enzyme centrifugation studies confirm that the monomer is a reactive species and suggest that the dimer also functions catalytically. Under the conditions of the usual enzyme assay, carbamoyl-phosphate synthetase is mainly in the monomer form. Activation by acetylglutamate can occur at the level of the monomer and is not coupled to dissociation since the enzyme dissociates at low concentrations even in the absence of acetylglutamate. The stoichiometry of the association is observed directly in the electron microscope. The dimensions of the negatively stained particles of the enzyme in the presence or absence of substrates correspond to monomers and dimers, assuming the molecule to be a prolate ellipse. The number of monomers observed in the presence of substrate represents 86% of the total number of enzyme molecules. The average molecular weight calculated from the numbers of particles seen in negatively stained specimens of carbamoyl-phosphate synthetase is 182000. Electron microscope studies provide independent evidence for monomer--dimer interactions and show that under the conditions examined the enzyme is mainly in the monomer form.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D002850:Chromatography, Gel; D005971:Glutamates / Q000378:metabolism; D008025:Ligases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008970:Molecular Weight; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D014461:Ultracentrifugation
D005971:Glutamates; D046911:Macromolecular Substances; D000244:Adenosine Diphosphate; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); C016195:N-acetylglutamic acid