CTP synthetase from Escherichia coli: an improved purification procedure and characterization of hysteretic and enzyme concentration effects on kinetic properties.
Previous studies have shown that CTP synthetase exists as a dimer which aggregates to a tetramer in the presence of the substrates ATP and UTP [Long, C. W., Levitzki, A., & Koshland, D. E., Jr. (1970) J. Biol. Chem. 245, 80]. A new, relatively simple purification procedure resulting in enzyme of high purity and in good yield has been established by using two successive hydrophobic chromatography steps, the first in the absence of ATP and UTP (dimer binds) and the second in the presence of ATP and UTP (tetramer does not bind). Several previously unreported properties of CTP synthetase are described which suggest that alterations in the state of association and dissociation of the enzyme have a controlling influence on the observed kinetic properties of the enzyme. The specific activity of CTP synthetase decreases with decreasing enzyme concentration, particularly when the concentrations of ATP and UTP in the reaction mixture are nonsaturating. The concentration of ATP or UTP required for half-maximal activity is significantly increased as the concentration of enzyme in the reaction mixture is decreased. CTP synthetase displays reversible cold lability and hysteretic properties (lags or bursts in the time course of product formation), both of which are influenced by the concentration of enzyme and/or the presence of ATP and UTP in the preincubation mixture and/or assay mixture. Gel filtration studies have shown that CTP synthetase can dissociate to an apparently inactive monomer. The dissociation is reversible, and the rate of association is slow.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D019731:Carbon-Nitrogen Ligases; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008025:Ligases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D014544:Uridine Triphosphate / Q000378:metabolism
D000255:Adenosine Triphosphate; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase; D014544:Uridine Triphosphate