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Labelling and exploration of the active site of enkephalinase ec 3.4.24.11 in kidney membranes with tritiated thirophan as ligand



Labelling and exploration of the active site of enkephalinase ec 3.4.24.11 in kidney membranes with tritiated thirophan as ligand



European Journal of Pharmacology 149(1-2): 121-130



[3H]Thiorphan, a potent inhibitor of enkephalinase (EC 3.4.24.11), was used to label the enzyme in membranes from rat kidney cortex and 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 .apprx. 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: the hydrophobic pocket in P1', the requirements of which are best satisfied by aromatic amino acid side chain residues, the P2' 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.

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