title: Thymidylate synthetase from Escherichia coli K12. Purification, and dependence of kinetic properties on sugar conformation and size of the 2' substituent.
abstract: Thymidylate synthetase from Escherichia coli K12 has been purified 3600-fold by a series of chromatographic procedures. The final preparation had a specific activity of 1.47 units/mg protein and was approximately 80% pure. The enzyme is a dimer of relative molecular mass, Mr, 64000 composed of two subunits of Mr 32,000 each. Its isoelectric point is 4.7 and it is stimulated by Mg2+. Michaelis constants for (+)5,10-methylene-5,6,7,8-tetrahydrofolate [(+)CH2H4folate] were 0.014 mM in the case of methylation of 2'-deoxyuridine-5'-phosphate (dUMP) and 0.55 mM when it served as methyl-group donor for 2'-fluoro-2'-deoxyuridine-5'-phosphate (dUflMP); the corresponding Km values for dUMP and dUflMP were 0.01 mM and 0.11 mM, respectively. The activation energies for the two reactions were found to be 72.8 kJ/mol (methylation of dUMP) and 66.1 kJ/mol (methylation of dUflMP). The data support a recognition mechanism between thymidylate synthetase and that fraction of the nucleotide the sugar moiety of which is in the 2'-endo-3'-exo conformation.
mesh_terms: D002236:Carbohydrate Conformation; D002845:Chromatography; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008780:Methyltransferases / Q000302:isolation & purification*; D008970:Molecular Weight; D013329:Structure-Activity Relationship; D013696:Temperature; D013940:Thymidylate Synthase / Q000302:isolation & purification*
chemical_list: D046911:Macromolecular Substances; D008780:Methyltransferases; D013940:Thymidylate Synthase