1-Phenyl-3-trimethylaminopropyl carbodiimide: a new inhibitor of thymidylate synthase

Thymidylate synthase (EC 2.1.1.45) from methotrexate-resistant Lactobacillus casei was inactivated by 1-phenyl-3-trimethylaminopropyl carbodiimide (PTC), 1-phenyl-3-dimethyl aminopropyl carbodiimide (PDC), and 1-ethyl-3-dimethyl aminopropyl carbodiimide (EDC). In the presence of excess PTC, the inactivation followed pseudo-first order kinetics; the second order rate constant was approximately 200 M-1min-1 at 30 degrees C. The rate of inactivation by PTC was faster than that by either PDC or EDC. Concentrations of the substrate dUMP greater than 0.15 mM, or of the product dTMP greater than 1.6 mM completely protected the enzyme from inactivation by PTC, but 10 mM dUrd provided very little protection. The rate of inactivation of EDC was reduced by only 40% in the presence of 50 mM dUMP. Nucleophiles (sulfanilic acid, glycine methyl ester, or glycine ethyl ester) had no effect on the rate of inactivation by PTC. The complete inactivation of thymidylate synthase by PTC was accompanied by the incorporation of approximately 2 mols of 14C-PTC per mol of enzyme. Although carbodiimides normally modify carboxyl groups in proteins, results from sulfhydryl group titrations and from reversible modification of sulfhydryl groups by methyl methanethiosulfonate suggest that two of the four cysteine residues of thymidylate synthase were modified by PTC.