Studies of 5-fluorodeoxyuridine 5'-monophosphate binding to carboxypeptidase A-inactivated thymidylate synthase from Lactobacillus casei.
The binding of 5-fluorodeoxyuridylate (FdUMP) to carboxypeptidase-inactivated thymidylate synthase obtained from methotrexate-resistant Lactobacillus casei was investigated using [3H]FdUMP in a trichloroacetic acid precipitation assay and by 19F nuclear magnetic resonance spectroscopy. The cleavage of 1 valine residue from the carboxyl terminus of one of the identical subunits of the enzyme dimer correlates with complete loss of thymidylate synthesis (Aull, J. L., Loeble, R. B., and Dunlap, R. B. (1974) J. Biol. Chem. 249, 1167-1172). We have further investigated the phenomenon of carboxypeptidase A-dependent inactivation of thymidylate synthase by employing immobilized carboxypeptidase A in order to facilitate the isolation and characterization of the inactivated enzyme. The time course of carboxypeptidase treatment of thymidylate synthase has been profiled by the spectrophotometric assay, tritium release assay, trichloroacetic acid precipitation assay (covalent adduct analysis), 19F nuclear magnetic resonance spectroscopy, and amino acid analysis. The techniques utilized in this study yielded results which showed that the completely inactivated enzyme (failure to catalyze thymidylate formation) continued to catalyze both covalent FdUMP-enzyme interactions and the formation of the covalent inhibitory ternary complex with the cofactor, 5,1O-methylenetetrahydrofolate, although to a reduced extent, thus effectively uncoupling these processes from thymidylate synthesis activity.
Cisneros
R J
RJ
Department of Chemistry and Biochemistry, University of South Carolina, Columbia 29208.
Zapf
J W
JW
Dunlap
R B
RB
D001665:Binding Sites; D002268:Carboxypeptidases / Q000378:metabolism*; D043422:Carboxypeptidases A; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005468:Fluorodeoxyuridylate / Q000378:metabolism*; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D013763:Tetrahydrofolates / Q000378:metabolism / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D004800:Enzymes, Immobilized; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D005468:Fluorodeoxyuridylate; D005492:Folic Acid; D013940:Thymidylate Synthase; D002268:Carboxypeptidases; D043422:Carboxypeptidases A