Mapping the active site of angiotensin-converting enzyme by transferred NOE spectroscopy.

The interaction of five furylacryloyl (fa)-amino acid derivatives, fa-Phe, fa-Phe-Phe, fa-Gly-Leu-NH(2), fa-Ala-Lys, and fa-Trp, with angiotensin-converting enzyme (ACE), a protein of MW = 130 kDa, was studied by transferred NOESY experiments. Identification of fa derivatives binding to ACE as well as determination of their relative affinities could be accomplished directly from the compound mixtures. Of the five fa derivatives we found that fa-Phe, fa-Trp, and fa-Gly-Leu-NH(2) bind more strongly to ACE than the other two. The dissociation constant of fa-Phe was determined from NMR spectra to 5 x 10(-4) M. A large excess of dipeptides competitively displaced fa-Trp and fa-Phe-Phe from the receptor pocket, allowing the binding site to be mapped. Also, the relative affinities of the fa-Phe, fa-Ala-Lys, and fa-Gly-Leu-NH(2) changed after addition of the dipeptides with fa-Gly-Leu-NH(2) showing the strongest binding. In addition, the presence of a strong inhibitor of the S1' and S2' sites, namely captopril, resulted in the same transferred NOE intensities of fa-Phe, indicating that it binds solely to the S1 and S2 subsites. A rapid screening of binding specificity from mixtures is possible by using a large excess of ligand(s) in transferred NOE studies, even when relatively small amounts of protein are present.