In a series of complexes of Lactobacillus casei dihydrofolate reductase (DHFR) formed with substrates and substrate analogues, the H-1/N-15 NMR chemical shifts for the guanidino group of the conserved Arg 57 residue were found to be sensitive to the mode of binding of their H-eta protons to the charged oxygen atoms in ligand carboxylate groups. In all cases, Arg 57 showed four nonequivalent H-eta signals indicating hindered rotation about the N-epsilon-C-zeta and C-zeta-N-eta bonds. The H-eta 12 and H-eta 22 protons have large downfield shifts as expected for a symmetrical end-on interaction with the ligand carboxylate group. The chemical shifts are essentially the same in the complexes with folate and p-aminobenzoyl-L-glutamate (PABG) and similar to those found previously for the methotrexate complex reflecting the strong and similar hydrogen bonds formed with the carboxylate oxygens. Interestingly, the rates of rotation about the N-epsilon-C-zeta bond for the complexes containing the weakly binding PABG fragment are almost identical to those measured in the complex with methotrexate, which binds 10(7) times more tightly. In the methotrexate complex, this rotation depends on correlated rotations about the N-epsilon-C-zeta bond of Arg 57 and the C-alpha-C' bond of the ligand glutamate cl-carboxylate group. Thus, even in a fragment such as PABG, which has a much faster off-rate, the carboxylate group binds to the enzyme in a similar way to that in a parent molecule such as folate and methotrexate with the rotation about the N-epsilon-C-zeta bond of Arg 57 being essentially the same in all the different complexes.
