Chen D H, Daron H H, Aull J L
Department of Chemistry, Auburn University, AL 36849-5312.
J Enzyme Inhib. 1992;5(4):259-68. doi: 10.3109/14756369109069068.
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.
来自对甲氨蝶呤耐药的干酪乳杆菌的胸苷酸合成酶（EC 2.1.1.45）可被1-苯基-3-三甲基氨基丙基碳二亚胺（PTC）、1-苯基-3-二甲基氨基丙基碳二亚胺（PDC）和1-乙基-3-二甲基氨基丙基碳二亚胺（EDC）灭活。在过量PTC存在的情况下，失活遵循假一级动力学；在30℃时二级速率常数约为200 M⁻¹min⁻¹。PTC导致的失活速率比PDC或EDC的都要快。底物dUMP浓度大于0.15 mM，或产物dTMP浓度大于1.6 mM时，可完全保护该酶不被PTC灭活，但10 mM的脱氧尿苷提供的保护作用很小。在50 mM dUMP存在的情况下，EDC的失活速率仅降低40%。亲核试剂（对氨基苯磺酸、甘氨酸甲酯或甘氨酸乙酯）对PTC导致的失活速率没有影响。PTC使胸苷酸合成酶完全失活的同时，每摩尔酶大约掺入2摩尔的¹⁴C-PTC。尽管碳二亚胺通常会修饰蛋白质中的羧基，但巯基滴定结果以及甲硫代磺酸甲酯对巯基的可逆修饰结果表明，胸苷酸合成酶的四个半胱氨酸残基中有两个被PTC修饰。
