New enkephalinase inhibitors as probes to differentiate “enkephalinase” and angiotensin-converting-enzyme active sites

Abstract

Thiorphan, N-[(R,S)-3-mercapto-2-benzylpropanoyl]-glycine the first described inhibitor (1) of the peptidyldipeptide hydrolase “enkephalinase” (2) was rationally synthesized taking into account the occurence in the Zn metallopeptidase of a S 1 ′ subsite specific for aromatic or hydrophobic residues (3,4), a feature lacking in angiotensin-converting-enzyme (ACE). In this work, we report new results concerning the differences between “enkephalinase” and ACE at the level of : i) their S 1 and catalytic subsites, ii) the interaction of inhibitors with the putative arginine residue located in both enzymes active site. For such a purpose, inhibitory potency (IC 50) of peptides, thiol and carboxy inhibitors (5) were measured on “enkephalinase” and ACE activities from mouse striatum. The 3 fold decreased potency of Leu-Phe-Leu as compared to Phe-Leu on “enkephalinase” suggest the absence of a hydrophobic S 1 subsite in this enzyme while such a lipophilic pocket occurs in ACE (6). Esterification or amidification of Thiorphan leads to a 4 to 10 times lower activity, a result also found in ACE but with a higher efficiency (60). The coordination of a COOH group to the Zn atom of ACE is strongly dependent of a right alignment of this group with the metal cation (7), a requirement not so stringent in “enkephalinase”. Therefore, this leads to mixed inhibitors as carboxymethyl Phe-Leu which acts as competitive inhibitor on both enzyme with IC 50 ∼ 2μM. By contrast, N-[(R,S)-2-carboxy, 3-benzylpropanoyl]-L-Leucine is a potent, K I = 0.34μM, competitive and highly selective (∼ 10,000 fold) “enkephalinase” inhibitor. On the other hand, routine dosage of “enkephalinase” activity requires a method without the time-consuming use of radioactive substrates as [ 3H]-Leu-E. In this way, we propose a simple method based on the large increase of fluorescence following the breakdown of the Gly-pNO 2Phe bond of dansyl-D-Ala-Gly-pNO 2Phe-Gly substrate by “enkephalinase”. Kinetic constants for this compound are K M = 20 μM, V m = 6.5 nmol/mg/prot/min, and K M = 11 μM, V m = 1.6 nmol/mg/prot/min, for its amidated derivative. Together with IC 50 values of the thiol inhibitors, these results suggest that brain “enkephalinase” behaves as an endopeptidase although exhibiting a preference for peptides with a free C-terminal carboxyl group. Finally, in order to investigate turn-over and sub-localization of “enkephalinase”, a photoaffinity label, azidothiorphan, was synthesized. Preliminary results show a covalent binding of this compound on “enkephalinase” in vitro and in vivo.