THYMIDYLATE SYNTHETASE FROM ESCHERICHIA-COLI-K12 - PURIFICATION, AND DEPENDENCE OF KINETIC-PROPERTIES ON SUGAR CONFORMATION AND SIZE OF THE 2' SUBSTITUENT

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 32000 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. Copyright © 1979, Wiley Blackwell. All rights reserved