Determination of subunit dissociation constants in native and inactivated CTP synthetase by sedimentation equilibrium.
Sedimentation equilibrium was used to correlate changes in aggregation state with active site modification of Escherichia coli CTP synthetase. The native enzyme equilibrated between monomers, dimers, and tetramers in the absence of substrates. At enzyme concentrations above 5 microM, tetramers represented 40% of the species in solution. Inactivation by 6-diazo-5-oxonorleucine (DON) or thiourea dioxide reduced the amount of tetramer to below detectable limits. However, inactivated enzyme still equilibrated between monomers and dimers. Simultaneous analysis of multispeed data at three protein concentrations yielded estimates of the dissociation constants for the monomer-dimer and dimer-tetramer equilibria. For multiple data sets of native enzyme, K1,2 was between 1 and 2 microM, and K2,4 was between 1 and 18 microM. For DON inactivated enzyme, K1,2 was 3-4 microM, and for thiourea dioxide inactivated enzyme, K1,2 was approximately 1 microM. The values for K1,2 are consistent with previously published studies by gel filtration, demonstrating that the enzyme dissociates to monomers in very dilute solution (Anderson, 1983). However, the sedimentation equilibrium experiments are the first to show that the enzyme forms tetramers in the absence of nucleotides. This result implies the presence of stable conformations in the native enzyme capable of dynamic equilibrium between monomers, dimers, and tetramers. The results presented here illustrate the sensitivity of sedimentation equilibrium for measuring the aggregation state of equilibrating enzyme species and demonstrate that active site modifications disrupt the quaternary structure of CTP synthetase.
Robertson
J G
JG
Enzymology Laboratory, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.
D000818:Animals; D019731:Carbon-Nitrogen Ligases; D002417:Cattle; D003980:Diazooxonorleucine / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008962:Models, Theoretical; D008970:Molecular Weight; D013816:Thermodynamics; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology
D046911:Macromolecular Substances; D003980:Diazooxonorleucine; C047256:thiourea dioxide; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase; D013890:Thiourea