Angiostrongylus cantonensis: characterization of thymidylate synthetase.
Thymidylate synthetase (TS) is the only enzyme that catalyzes the formation of thymidine nucleotides in Angiostrongylus cantonensis. A fraction enriched in TS was obtained from the gravid nematode by gel filtration and affinity chromatography using methotrexate-agarose. TS, which was well separated from dihydrofolate reductase, has a relative molecular mass of 66 kDa. By electrophoresis in sodium dodecyl sulphate gel, a major protein band corresponding to 31 kDa was observed. This band was shown to be TS by comparing the electrophoretic mobility with an enzyme preparation bound with [6-3H]5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). Therefore, the enzyme is composed of two identical or very similar subunits. Velocity studies and product inhibition patterns revealed that the TS reaction undergoes a sequential mechanism in which 2'-deoxyuridine 5'-monophosphate (dUMP) is the first substrate added to the active site and thymidine 5'-monophosphate is the last product released. The apparent Km values for dUMP and 5,10-methylenetetrahydrofolate are 10 and 185 microM, respectively. FdUMP and trimethoprim inhibited the parasite TS competitively with dUMP and the Ki values of 23.5 nM and 852 microM, respectively. Methotrexate was a noncompetitive inhibitor of TS. At 0.2 mM 5,10-methylenetetrafolate, 1 mM methotrexate inhibited the activity by 74%.
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Department of Biochemistry, University of Hong Kong.
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D000645:Ammonium Sulfate; D017185:Angiostrongylus cantonensis / Q000201:enzymology*; D000818:Animals; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology; D013763:Tetrahydrofolates / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D014295:Trimethoprim / Q000494:pharmacology
D003856:Deoxyuracil Nucleotides; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; C007267:2'-deoxyuridylic acid; D014295:Trimethoprim; D013940:Thymidylate Synthase; D000645:Ammonium Sulfate; D008727:Methotrexate