Propioxatins A and B, New Enkephalinase B Inhibitors. IV. Characterization of the Active Site of the Enzyme Using Synthetic Propioxatin Analogues

Abstract

Propioxatins A and B are inhibitors of enkephalinase B, which hydrolyzes enkephalin at the Gly-Gly bond. In order to clarify the structure-activity relationships of propioxatin, several compounds were synthesized and their inhibitory activity for not only enkephalinase B but also enkephalinae A was examined. The hydroxamic acid group in propioxatin was primarily essential for coordinating the metal ion in the active site of the enzyme. Among devalyl propioxatin A derivatives, the proline-containing compounds inhibited enkephalinase B and others inhibited both enzymes. An alteration of the character of the P₃', amino acid valine in propioxatin A, e.g. amidation of carboxylic acid or replacement of the side chain, caused a 2 to 400-fold decrease of the inhibitory activity for enkephalinase B or an appearance of enkephalinase A inhibition with K₁ values in the micromolar range. Substitution of the proline by alanine also resulted in a 1, 000-fold loss of inhibitory activity for enkephalinase B. Propioxatin A was the most potent and specific inhibitor of enkephalinase B among the synthesized compounds. These potent and specific inhibitory effects were caused by the P₂' proline residue, the P₃' valine side chain and its free carboxylic acid. Each of the S₁', S₂', and S₃' subsites in an enkephalinase B active site has a large and hydrophobic pocket, but the arrangement might be unique. The results could explain why enkephalinase B does not hydrolyze longer peptides.