PMID: 42538Dec 1, 1979

Thymidylate synthetase from Escherichia coli K12. Purification, and dependence of kinetic properties on sugar conformation and size of the 2' substituent

European Journal of Biochemistry
T HaertléW Guschlbauer

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.

References

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Citations

Mar 15, 1977·Biochimica Et Biophysica Acta·K A Chalabi, W E Gutteridge
Apr 19, 1975·Biochimica Et Biophysica Acta·R W McCuen, F M Sirotnak
Aug 1, 1976·Analytical Biochemistry·F Iborra, J M Buhler
Oct 1, 1973·Archives of Biochemistry and Biophysics·G R CapcoC K Mathews
Jan 1, 1972·Advances in Enzyme Regulation·M Friedkin, E Donovan
Jan 5, 1971·Biochemistry·R B DunlapF M Huennekens
Dec 1, 1967·Tetrahedron Letters·M YoshikawaT Takenishi
Feb 1, 1962·Proceedings of the National Academy of Sciences of the United States of America·G R GREENBERGS DEWOLF

Related Concepts

Thymidylate Synthase
Alkalescens-Dispar Group
Structure-Activity Relationship
Chromatography
Carbohydrate Linkage
TYMS
Methyltransferase
Hydrogen-Ion Concentration

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