Characterisation of neprilysin (EC 3.4.24.11) S2′ subsite

Abstract
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 non-mutated and mutated enzymes with dipeptide inhibitors and hydrolysis of the substrate [Leu5, Arg6]enkephalin in order to evaluate the contribution of the S2′ 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 P2′ residue of the substrate, (2) the S2′ subsite is vast and can accommodate bulky side chains, and (3) Arg-102 restricts access to the S2′ subsite to some side chains such as arginine.