Abstract
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.
Highlights
Thymidylate synthetase of Saccharomyces cerevisiae proliferatingcells, such as in embryonictissues, generallyhave was purified over 20,000-fold to apparenthomogeneity elevated levels of thymidylate synthetase [3, 4]
Halogen- bakers’ yeast.we have shown previously [17] that ated analogs of the nucleotide substrate were compet- cdc21, a locusoriginallydefinedbya heat-sensitive condiitive inhibitors of the yeast enzyme, with apparent Ki tional-lethal mutation which caused arrest of the yeast cell values for 5-fluoro-dUMPof 5 n~ and for 5-Br-dUMPof division cycle during S phase [18],is the structural
Inhibitors, with apparent Ki values for both methotrex- Yeast cells appear tobe similar tohigher eukaryotes in the ate and aminopterin of about 20 p ~ C.ibacron blue, a respect that a number of functions involved in deoxyribonudye used as the ligand in an affinity adsorbent forone cleotide metabolism have been reported todisplay maximum of the purification steps, was a potentcompetitive in- activity and/or rate of synthesis during S phase, including hibitor with respect to either substratey, ielding apparent Ki values of 4 nM for thenucleotide binding site and 40 xm for the cofactor binding site
Summary
From the Departmentof Microbiology and Immunology, University of California, Berkeley, California 94720. ’ methyltransferase, EC 2.1.1.45) catalyzes methylation of the ease of genetic and biochemical manipulation of yeast makes pyrimidine ringof dUMP, yielding dTMP In most eukaryotic this organism attractive forinvestigating regulation of the cells the level of thymidylate synthetase liosw, and production expression and activityof thymidylate synthetase in a eukarof dTMP by the enzyme is believed to be rate limiting for yotic cell. To begin such a study itwas essential to determine.
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