Thermodynamic study of the influence of NADPH on the binding of methotrexate and its metabolites to a mammalian dihydrofolate reductase.
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 pK 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 of its enzymatic activity.
Gilli
R M
RM
Laboratoire de Physique Pharmaceutique, Faculté de Pharmacie, Marseille, France.
Sari
J C
JC
Sica
L M
LM
Briand
C M
CM
D000818:Animals; D002151:Calorimetry; D002417:Cattle; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D008727:Methotrexate / Q000031:analogs & derivatives / Q000378:metabolism*; D009249:NADP / Q000494:pharmacology*; D010084:Oxidation-Reduction; D011199:Potentiometry; D011485:Protein Binding; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate