Thymidylate synthetase from Saccharomyces cerevisiae. Purification and enzymic properties.
Thymidylate synthetase of Saccharomyces cerevisiae was purified over 20,000-fold to apparent homogeneity by a procedure involving two new affinity methods and several precautions for avoiding proteolysis. Molecular weight of the native enzyme was about 65,000, as determined by gel filtration and velocity sedimentation. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band of molecular weight 30,000, suggesting that thymidylate synthetase is a dimer of very similar or identical subunits. The purified enzyme exhibited normal Michaelis-Menten kinetics toward both substrates, with apparent Km values for dUMP and for (--)-5,10-methylene-tetrahydropteroylglutamate of 5 microM and 70 microM, respectively. When the pentaglutamyl form of the cofactor was used, its apparent Km was lower (7 microM), but Vmax was unaltered. Reaction kinetics and product inhibition studies were most consistent with an ordered mechanism, wherein dUMP is the first substrate to bind and 7,8-dihydropteroylglutamate is the first product released. Halogenated analogs of the nucleotide substrate were competitive inhibitors of the yeast enzyme, with apparent Ki values for 5-fluoro-dUMP of 5 nM and for 5-Br-dUMP of 10 microM. Analogs of the cofactor were also competitive inhibitors, with apparent Ki values for both methotrexate and aminopterin of about 20 microM. Cibacron blue, a dye used as the ligand in an affinity adsorbent for one of the purification steps, was a potent competitive inhibitor with respect to either substrate, yielding apparent Ki values of 4 nM for the nucleotide binding site and 40 nM for the cofactor binding site.
D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008780:Methyltransferases / Q000302:isolation & purification*; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013940:Thymidylate Synthase / Q000302:isolation & purification* / Q000378:metabolism
D046911:Macromolecular Substances; D008780:Methyltransferases; D013940:Thymidylate Synthase