Methotrexate binding

Abstract: The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s(-1) and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.

Abstract: The crystal structure of recombinant human dihydrofolate reductase with folate bound in the active site has been determined and the structural model refined at 0.2-nm resolution. Preliminary studies of the binding of the inhibitors methotrexate and trimethoprim to the human apoenzyme have been performed at 0.35-nm resolution. The conformations of the chemically very similar ligands folate and methotrexate, one a substrate the other a potent inhibitor, differ substantially in that their pteridine rings are in inverse orientations relative to their p-aminobenzoyl-l-glutamate moieties. Methotrexate binding is similar to that previously observed in two bacterial enzymes but is quite different from that observed in the enzyme from a mouse lymphoma cell line [Stammers et al. (1987) FEBS Lett. 218, 178–1841. The geometry of the polypeptide chain around the folate binding site in the human enzyme is not consistent with conclusions previously drawn with regard to the species selectivity of the inhibitor trimethoprim [Matthews et al. (1985) J. Biol. Chem. 260, 392–399].

Abstract: Information was sought on the relative extent to which transport-defective, methotrexate-resistant phenotypes emerge among the total subpopulation of resistant phenotypes during therapeutic challenge of leukemic cells in vivo. A number of monoclonal methotrexate-resistant sublines of the L1210 leukemia were derived during methotrexate therapy of leukemic mice and biochemically characterized. Of the total number of 14 sublines derived, five exhibited altered [3H]methotrexate transport alone, five exhibited increased dihydrofolate reductase content alone (2- to 18-fold), and four showed alterations in both of these properties. Methotrexate binding and substrate turnover rate for dihydrofolate reductase appeared to be unchanged in any of the resistant sublines. The relative resistance of each subline was accounted for by the biochemical alterations observed. Among the transport-defective sublines, one subcategory showed a 3- to 4-fold reduction in apparent influx Vmax for [3H]methotrexate, a second category showed both a 5-fold reduction in influx Vmax and a 3-fold increase in the apparent influx Km, and one subline showed only a 2-fold increase in Km. Otherwise, Michaelis-Menten saturation kinetics for influx was observed in each case and in the case of the parental line and the other resistant sublines. None of the resistant sublines exhibited altered efflux of [3H]methotrexate. Steady-state levels measured for intracellular exchangeable (osmotically active) fractions of drug accurately reflected the values for specific kinetic parameters determined for each sensitive and resistant cell line. These studies show that transport-defective phenotypes represent a major category of methotrexate-resistant cell types which emerge initially from leukemic cell populations under therapy in mice. Based on considerations discussed here, it is reasonable to assume that a similar relative occurrence of this phenotype would result during methotrexate therapy of leukemia patients.