Nuclear magnetic resonance studies of the binding of trimethoprim to dihydrofolate reductase

Abstract

The resonances of the aromatic protons of trimethoprim [an effective antibacterial agent] [2,4-diamino-5-(3'',4'',5''-trimethoxybenzyl)pyrimidine] in its complexes with dihydrofolate reductases (EC 1.5.1.3) from Lactobacillus casei and Escherichia coli cannot be directly observed. Their chemical shifts were determined by transfer of saturation experiments and by difference spectroscopy using [2'',6''-2H2]trimethoprim. The complex of 2,4-diamino-5-(3'',4''-dimethoxy-5''-bromobenzyl)pyrimidine with the L. casei enzyme was also examined. At room temperature, the 2'',6''-proton resonance of bound trimethoprim is very broad (line width > 30 Hz); with the E. coli enzyme, the resonance sharpens with increasing temperature so as to be clearly visible by difference spectroscopy at 45.degree. C. This line broadening is attributed to an exchange contribution, arising from the slow rate of flipping about the C7.sbd.C1'' bond of bound trimethoprim. The transfer of saturation measurements were also used to determine the dissociation rate constants of the complexes. In the course of these experiments, a decrease in intensity of the resonance of the 2'',6''-proton resonance of free trimethoprim on irradiation at the resonance of the 6 proton of free trimethoprim was observed, which only occurred in the presence of the enzyme. This is interpreted as a nuclear Overhauser effect between 2 protons of the bound ligand transferred to those of the free ligand by the exchange of the ligand between the 2 states. The chemical shift changes observed on the binding of trimethoprim to dihydrofolate reductase are interpreted in terms of the ring-current shift contributions from the 2 aromatic rings of trimethoprim and from that of phenylalanine-30. On the basis of this analysis of the chemical shifts, a model for the structure of the enzyme-trimethoprim complex is proposed. This model is consistent with the (indirect) observation of a nuclear Overhauser effect between the 2'',6'' and 6 protons of bound trimethoprim.