CALCULATION OF THE REACTION PATHWAY FOR THE AROMATIC RING FLIP IN METHOTREXATE COMPLEXED TO DIHYDROFOLATE-REDUCTASE

The rotation of the benzoyl ring of methotrexate has been modeled in i ts complexes with dihydrofolate reductase (DHFR). The conjugate peak r efinement method (Fischer, S.; Karplus, M. Chem. Phys. Lett. 1992, 194 , 252) was used to generate conformational reaction paths and to locat e transition states for the 180 degrees ring-flip process. The compute d energy barriers for the 3',5'-fluoro-substituted benzoyl ring of met hotrexate (F(2)MTX) are 11.3 and 10.1 kcal/mol for the binary and tern ary (with cofactor NADPH) complexes, respectively, which compare well with the experimental enthalpies of activation of 11.5 (binary) and 9. 9 kcal/mol (ternary) from F-19-nmr spectroscopy (Clore, G. M.; Gronenb orn, A. M.; Birdsall, B.; Feeney, J.; Roberts, G. C. K. Biochem. J. 19 84, 217, 659). The pathways for the hydrogen-substituted isomer (H(2)M TX) are found to be similar, although the computed barrier heights are lower (6.5 and 5.0 kcal/mol, respectively). The process is characteri zed by an asynchronous transition of the two dihedral angles adjacent to the benzoyl ring and by a twin gating of the ring flip by four resi dues (Leu27, Phe30, Phe49, and Pro50), which form a ''hydrophobic quad rant'' around the ring. Perturbations of the protein up to 8 Angstrom from the active site (which expands by 1.6 Angstrom) make contribution s to the energetics of the process. The local and global characteristi cs of the path and the effects of structural (crystallographic) solven t and the cofactor are discussed.