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

• Published in: Journal of biochemistry (Tokyo) Vol. 104; no. 5; pp. 706 - 711
• Main Authors: Inaoka, Yoshinori, Naruto, Shunji
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.11.1988
• Subjects: Analytical, structural and metabolic biochemistry; Binding Sites; Biological and medical sciences; Dipeptides - analysis; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - antagonists & inhibitors; Endopeptidases; enkephalinase B; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Models, Molecular; Neprilysin - antagonists & inhibitors; propioxatins; Protease Inhibitors; Structure-Activity Relationship; Characterization; Synthetic product; Investigation method; Analog; Endopeptidase; Active site; Enzyme inhibitor
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a122537

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.