CONSIDERATION OF THE PH-DEPENDENT INHIBITION OF DIHYDROFOLATE-REDUCTASE BY METHOTREXATE

Poisson-Boltzmann calculations were used to determine the pK(a) of pro tein functional groups in the unliganded dihydrofolate reductase enzym e, and the pK(a) of protein and ligand groups in methotrexate-enzyme c omplexes. The results reported here are in conflict with two fundament al tenets of dihydrofolate reductase inhibition by methotrexate: (1) A sp27 is not expected to be protonated near pH 6.5 in the apoenzyme as previously proposed based on fitting of empirical equations to binding data, and (2) the calculated pK(a) for the pteridine N1 of rite inhib itor while bound to the protein is significantly lower than that estim ated for this group from Interpretation of NMR data (>10). In fact, th e electrostatic calculations and complementary quantum chemical calcul ations indicate that Asp27 is likely protonated when methotrexate is b ound, resulting in a neutral dipole-dipole interaction rather than a s alt-bridge between the enzyme and the inhibitor. Reasons for this disc repancy with the experimental data are discussed. Furthermore, His45 a nd Glu17 in the Escherichia coli enzyme are proposed to be in part res ponsible for the pH dependence of the conformational degeneracy in the inhibitor-enzyme complex. (C) 1997 Academic Press.