Thermodynamic study of the influence of NADPH on the binding of methotrexate and its metabolites to a mammalian dihydrofolate reductase

• Published in: Biochimica et biophysica acta Vol. 964; no. 1; pp. 53 - 60
• Main Authors: Gilli, Robert M., Sari, Jean C., Sica, Lucas M., Briand, Claudette M.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 12.01.1988; Elsevier; North-Holland
• Subjects: 7-Hydroxymethotrexate; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Calorimetry; Cattle; Dihydrofolate reductase; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Kinetics; Liver - enzymology; Mathematics; Methotrexate - analogs & derivatives; Methotrexate - metabolism; Methotrexate binding; Methotrexate polyglutamate; NADP - pharmacology; NADPH; Oxidation-Reduction; Oxidoreductases; Potentiometry; Protein Binding; Tetrahydrofolate Dehydrogenase - metabolism; NADPH; Methotrexate polyglutamate; 7-Hydroxymethotrexate; methotrexate-Glu n; Methotrexate binding; Dihydrofolate reductase; methotrexate polyglutamate where n equals the number of additional glutamate residues; Thermodynamics; Vertebrata; Mammalia; Enzyme; Metabolite; Tetrahydrofolate dehydrogenase; Microcalorimetry; Potentiometry
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(88)90066-9

Interaction of methotrexate and some of its metabolites with a mammalian dihydrofolate reductase was studied using two complementary methods, potentiometry and microcalorimetry. The major plasma metabolite of this anticancer agent, 7-hydroxymethotrexate, was found to have a different binding behavior from that of polyglutamyl derivatives and of methotrexate itself. Indeed, 7-hydroxymethotrexate binds without a p K shift to dihydrofolate reductase, whereas polyglutamyl derivatives bind to the enzyme with a proton uptake, as the parent drug does. NADPH increases the association constant of the 7-hydroxy metabolite by a factor of 10–20, while for methotrexate and for polyglutamates this increase is about 100-fold. It was demonstrated that the enhancement of the binding by NADPH had an enthalpic origin. Finally, the binding behavior of dihydrofolate reductase seemed to be independent to its enzymatic activity.