Conformational analysis of an EP24.15 inhibitor by NMR and molecular modelling

The enzyme thimet oligopeptidase (EC3.4.24.15, EP24.15) is responsible for the hydrolysis of a number of neuropeptides. Despite much research examining its substrate specificity, little is known about the conformational requirements of its active site. We have used 1D ¹H and 2D TOCSY NMR experiments to assign the proton resonances of the EP24.15 inhibitor, N-[1- (R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP), and 2D ROESY NMR to investigate whether cFP exhibits any conformational preferences in CD₃OD and in aqueous CD₃OD. Molecular modelling of charged cFP in the gaseous phase generated a number of conformations that were consistent with the NMR data obtained in CD₃OD. Analogous modelling on the uncharged cFP did not result in conformations consistent with any of the NMR data, but did suggest that, under non-polar conditions, cFP could adopt a hairpin conformation which would allow simultaneous coordination of the two carboxyl groups of cFP to the zinc ion in the active site of EP24.15.