Purification and characterization of the bifunctional thymidylate synthetase-dihydrofolate reductase from methotrexate-resistant Leishmania tropica.
Thymidylate synthetase (TS) and dihydrofolate reductase (DHFR) in Leishmania tropica exist as a bifunctional protein. By use of a methotrexate-resistant strain, which overproduces the bifunctional enzyme, the protein was purified 80-fold to apparent homogeneity in two steps. The native protein has an apparent molecular weight of 110 000 and consists of two subunits with identical size and charge. Available data indicate that each of the subunits possesses TS and DHFR. The TS of the bifunctional protein forms a covalent 5-fluoro-2'-deoxyuridylate (FdUMP)-(+/-)-5,10-methylenetetrahydrofolate-enzyme complex in which 2 mol of FdUMP is bound per mole of enzyme. In contrast, titration of DHFR with methotrexate indicated that only 1 mol of the inhibitor is bound per mole of dimeric enzyme. Both TS and DHFR activities of the bifunctional enzyme were inactivated by the sulfhydryl reagent N-ethylmaleimide. Substrates of the individual enzymes afforded protection against inactivation, indicating that each enzyme requires at least one cysteine for catalytic activity. Kinetic evidence indicates that most, if not all, of the 7,8-dihydrofolate produced by TS is channeled to DHFR faster than it is released into the medium. Although the mechanism of channeling is unknown, the possibility that the two enzymes share a common folate binding site has been ruled out.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004351:Drug Resistance; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D007891:Leishmania / Q000201:enzymology*; D046911:Macromolecular Substances; D008727:Methotrexate / Q000494:pharmacology*; D008780:Methyltransferases / Q000302:isolation & purification*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D013045:Species Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D013940:Thymidylate Synthase / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D013762:Tetrahydrofolate Dehydrogenase; D008780:Methyltransferases; D013940:Thymidylate Synthase; D008727:Methotrexate