UNUSUAL BINDING STOICHIOMETRIES AND COOPERATIVITY ARE OBSERVED DURINGBINARY AND TERNARY COMPLEX-FORMATION IN THE SINGLE ACTIVE PORE OF R67DIHYDROFOLATE-REDUCTASE, A D-2 SYMMETRICAL PROTEIN

R67 dihydrofolate reductase (DHFR) is an R-plasmid-encoded enzyme that confers resistance to the antibacterial drug, trimethoprim. This DHFR variant is not homologous in either sequence or structure to chromoso mal DHFRs. A recent crystal structure of the active tetrameric species describes a single active site pore that traverses the length of the protein (Narayana et al., 1995). Related sites (due to a 222 symmetry element at the center of the active site pore) are used for binding of ligands, i.e., each half-pore can accommodate either the substrate, d ihydrofolate, or the cofactor, NADPH, although dihydrofolate and NADPH are bound differently. Ligand binding in R67 DHFR was evaluated using time-resolved fluorescence anisotropy and isothermal titration calori metry techniques, Under binary complex conditions, two molecules of ei ther NADPH, folate, dihydrofolate, or N10 propargyl-5,8-dideazafolate (CB3717) can be bound, Binding of NADPH displays negative cooperativit y, binding of either folate or dihydrofolate shows positive cooperativ ity, and binding of CB3717 shows two identical sites. Any asymmetry in troduced by binding of one ligand is proposed to induce the cooperativ ity associated with binding of the second ligand, Evaluation of ternar y complex formation demonstrates that one molecule of folate binds to a 1:1 mixture of R67 DHFR + NADPH. These binding results indicate a ma ximum of two ligands bind in the pore. A mechanism describing catalysi s is proposed that is consistent with the binding results.