Inactivation and covalent modification of CTP synthetase by thiourea dioxide.
Thiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase. Incubation at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme. The second-order rate constant for inactivation was 0.18 M-1 s-1. Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed-function oxidation/reduction as the mode of amino acid modification. Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not. The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides. Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection. The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP. In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case. Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein. Modification with [14C]thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of [14C]thiourea dioxide per mole of CTP synthetase monomer. The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity. The data further indicate that nucleotide binding prevents access to these functionally important residues.
Robertson
J G
JG
Department of Chemistry, Pennsylvania State University, University Park 16802.
Sparvero
L J
LJ
Villafranca
J J
JJ
D001665:Binding Sites; D019731:Carbon-Nitrogen Ligases; D002942:Circular Dichroism; D004586:Electrophoresis; D005453:Fluorescence; D005973:Glutamine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008025:Ligases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D009711:Nucleotides / Q000378:metabolism; D013379:Substrate Specificity; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology
D009711:Nucleotides; D005973:Glutamine; C047256:thiourea dioxide; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase; D013890:Thiourea