1H and 15N NMR studies of protonation and hydrogen-bonding in the binding of trimethoprim to dihydrofolate reductase.

The binding of trimethoprim and [1,3,2-amino-15N3]-trimethoprim to Lactobacillus casei dihydrofolate reductase has been studied by 15N and 1H NMR spectroscopy. 15N NMR spectra of the bound drug were obtained by using polarisation transfer pulse sequences. The 15N chemical shifts and 1H-15N spin-coupling constants show unambiguously that the drug is protonated on N1 when bound to the enzyme. The N1-proton resonance in the complex has been assigned using the 15N-enriched molecule. The temperature-dependence of the linewidth of this resonance has been used to estimate the rate of exchange of this proton with the solvent: 160 +/- 10 S-1 at 313 K, with an activation energy of 75 (+/-9) kJ X mole-1. This is considerably faster than the dissociation rate of the drug from this complex, demonstrating that there are local fluctuations in the structure of the complex.