Neprilysin (EC 3.4.24.11) is a Zn2+ metallopeptidase involved in the degradation of biologically active peptides, e.g. enkephalins and atrial natriuretic peptide. The substrate specificity and catalytic activity of neprilysin resemble those of thermolysin, a crystallized bacterial Zn2+ metalloprotease. Despite little overall homology between the primary structures of thermolysin and neprilysin, many of the amino acid residues involved in catalysis, as well as Zn2+ and substrate binding, are highly conserved. Most of the active-site residues of neprilysin have their homologues in thermolysin and have been characterized by site-directed mutagenesis. Furthermore, hydrophobic cluster analysis has revealed some other analogies between the neprilysin and thermolysin sequences [Benchetrit, Bissery, Mornon, Devault, Crine and Roques (1988) Biochemistry 27, 592-596]. According to this analysis the role of Asn542 in the neprilysin active site is analogous to that of Asn112 of thermolysin, which is to bind the substrate. Site-directed mutagenesis was used to change Asn542 to Gly or Gln residues. The effect of these mutations on substrate catalysis and inhibitor binding was examined with a series of thiorphan-like compounds containing various degrees of methylation at the P2' residue. For both mutated enzymes, determination of kinetic parameters with [D-Ala2,Leu5]enkephalin as substrate showed that the large decrease in activity was attributable to an increase in Km (14-16-fold) whereas kcat values were only slightly affected (2-3-fold decrease). This is in agreement with Asn542 being involved in substrate binding rather than directly in catalysis. Finally, the IC50 values for thiorphan and substituted thiorphans strongly suggest that Asn542 of neprilysin binds the substrate on the amino side of the P2' residue by formation of a unique hydrogen bond.

Neutral endopeptidase 24.11 (EC 3.4.24.11; NEP) is a membrane-bound Zn-metalloendopeptidase with a catalytic activity and a specificity very similar to that of thermolysin, a bacterial zinc-endoprotease. NEP can be inactivated by reaction with diethylpyrocarbonate, due to the modification of a histidine residue present in the active site of the enzyme. This histidine residue was proposed to be analogous to His231 in thermolysin, which is involved in the stabilization of the tetrahedral intermediate during the transition state. Using site-directed mutagenesis of the cDNA encoding rabbit NEP, we have created two mutants of NEP where His711 was replaced by either Gln or Phe (NEP-Gln711 and NEP-Phe711). Determination of kinetic parameters showed that both mutants had Km values very similar to that of the non-mutated enzyme but that their kcat values were 25-fold lower. The calculated difference in free energy needed to form the transition state complex was increased by 2.2 kcal/mol for both mutants. These observations strongly suggest that His711 is involved in the stabilization of the transition state by forming an hydrogen bond with the oxyanion of the tetrahedral intermediate.

Neutral endopeptidase (NEP) is a membrane-bound mammalian ectopeptidase that contains a catalytic zinc ion in its active site. Previous studies showed that the active site, and especially the zinc-binding site of NEP, have features in common with the prototypical bacterial zinc protease, thermolysin. Sequence comparison reveals that both enzymes have a conserved Asp residue (Asp650 in NEP and Asp170 in thermolysin) located four positions on the C-side of the third zinc ligand. In thermolysin, this residue is involved in a carboxylate-histidine-zinc interaction whose functional role has never been established

Neprilysin is a neutral peptidase that cleaves small peptide substrates on the amino-side of hydrophobic amino acid residues. In the present study, we have used inhibition of nonmutated and mutated enzymes with dipeptide inhibitors and hydrolysis of the substrate [Leu 5 , Arg 6 ]enkephalin in order to evaluate the contribution of the S 2 ' subsite to substrate and inhibitor binding. Our results suggest that (1) Arg-102 and Asn-542 provide major contributions to the interaction of the enzyme with the P 2 ' residue of the substrate, (2) the S 2 ' subsite is vast and can accommodate bulky side chains, and (3) Arg-102 restricts access to the S 2 ' subsite to some side chains such as arginine.