Labelling and exploration of the active site of enkephalinase (EC 3.4.24.11) in kidney membranes with [3H]thiorphan as ligand.
[3H]Thiorphan, a potent inhibitor of enkephalinase (EC 3.4.24.11), was used to label the enzyme in membranes from rat kidney cortex an to explore its specificity at the active site. [3H]Thiorphan binding occurred reversibly, with low non-specific binding and to a single class of sites. The dissociation constant, determined by either kinetics or saturation studies was approximately 0.4 nM. The ratio of the maximal velocity of enkephalinase with enkephalins as substrates to the maximal binding of [3H]thiorphan was consistent with the catalytic constant of the enzyme. Enkephalinase inhibitors competed with [3H]thiorphan and had inhibitory constants in agreement with the corresponding values derived from measurement of the enzyme catalytic activity, whereas inhibitors of other metallopeptidases were ineffective. The inhibitory potencies of a series of systematically varied oligopeptides regarding [3H]thiorphan binding and enkephalinase activity were also highly correlated. Structure-activity relationships among competitors indicated that the main subsites of enkephalinase are: (1) the hydrophobic pocket in P'1, the requirements of which are best satisfied by aromatic amino acid side chain residues (2) the P'2 subsite, the requirements of which are best satisfied by amino acids with a short, uncharged side chain and a free terminal carboxyl group. This novel binding assay should facilitate the exploration of the active site of enkephalinase and the development of new inhibitors.
De la Baume
S
S
Unité 109 de Neurobiologie et Pharmacologie, Centre Paul Broca de l'INSERM, Paris, France.
Schwartz
J C
JC
D000603:Amino Acids, Sulfur; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology*; D008024:Ligands; D008297:Male; D008666:Metalloendopeptidases / Q000378:metabolism*; D015260:Neprilysin; D011480:Protease Inhibitors; D051381:Rats; D011919:Rats, Inbred Strains; D015244:Thiorphan; D008625:Tiopronin / Q000031:analogs & derivatives
D000603:Amino Acids, Sulfur; D008024:Ligands; D011480:Protease Inhibitors; D015244:Thiorphan; D008625:Tiopronin; D008666:Metalloendopeptidases; D015260:Neprilysin