Thymidylate Synthetase from Escherichia coli K12. Purification, and Dependence of Kinetic Properties on Sugar Conformation and Size of the 2' Substituent

Open Access
Publisher Website
Google Scholar
Abstract
Thymidylate synthetase from E. coli K12 was 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, 64,000 composed of two subunits of Mr 32,000 each. Its isoelectric point is 4.7 and it is stimulated by Mg2+. Km for (+)5,10-methylene-5,6,7,8-tetrahydrofolate [(+)CH2H4folate] were 0.014 mM in the case of methylation of dUMP and 0.55 mM when it served as methyl-group donor for 2''-fluoro-dUMP (dUflMP); the corresponding Km values for dUMP and dUflMP were 0.01 and 0.11 mM, respectively. The activation energies for the 2 reactions were 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.