Presence and properties of thymidylate synthase in trypanosomatids.
High speed centrifugal supernatant fractions of homogenates of a number of trypanosomatids were assayed for thymidylate synthase (5,10-methylene-tetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) activity using the method of Lomax and Greenberg (1967) J. Biol. Chem. 242, 109-113). Similar activities were detected in Crithidia fasciculata, Crithidia oncopelti, the blood forms of Trypanosoma brucei, Trypansoma congolense and Trypanosoma lewisi and the blood, intracellular and culture forms of Trypanosoma cruzi, suggesting that all species synthesize at least some thymidylate de novo. The properties of the activities in C. fasciculata and the three forms of T. cruzi were compared with those of the isofunctional bacterial and mammalian enzymes. The trypanosotamid enzyme was inhibited by Mg2+, was much more sensitive to mercaptoethanol, had higher apparent Km values for substrate (dUMP) and cofactor (tetrahydrofolate), had a higher apparent molecular weight and was markedly more sensitive to inhibition by suramin. It is, therefore a possible target for chemotherapeutic attack, either on its own or in combination with a dihydrofolate reductase inhibitor. No evidence was obtained for the regulation of the trypanosomatid enzyme, either by its product, dTMP, or by dTDP or dTTp. This result agrees with previous studies which suggested that in trypanosomatids, the level of dTMP was regulated, at least in part, by a catabolic pathway consisting of a thymidylate phosphatase and a thymidine phosphorylase which degraded the excess of dTMP to thymine.
D000818:Animals; D056890:Eukaryota / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008780:Methyltransferases / Q000378:metabolism*; D008970:Molecular Weight; D013045:Species Specificity; D013498:Suramin / Q000494:pharmacology; D013696:Temperature; D013940:Thymidylate Synthase / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000494:pharmacology; D014345:Trypanosoma / Q000201:enzymology*; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D014349:Trypanosoma cruzi / Q000201:enzymology*; D014350:Trypanosoma lewisi / Q000201:enzymology*
D013942:Thymine Nucleotides; D013498:Suramin; D008780:Methyltransferases; D013940:Thymidylate Synthase