EVIDENCE THAT ASN(542) OF NEPRILYSIN (EC-3.4.24.11) IS INVOLVED IN BINDING OF THE P-2' RESIDUE OF SUBSTRATES AND INHIBITORS

Neprilysin (EC 3.4.24.11) is a Zn2+ metallopeptidase involved in the d egradation of biologically active peptides, e.g. enkephalins and atria l natriuretic peptide. The substrate specificity and catalytic activit y of neprilysin resemble those of thermolysin, a crystallized bacteria l Zn2+ metalloprotease. Despite little overall homology between the pr imary 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 s ite-directed mutagenesis. Furthermore, hydrophobic cluster analysis ha s revealed some other analogies between the neprilysin and thermolysin sequences [Benchetrit, Bissery, Mornon, Devault, Crine and Rogues (19 88) Biochemistry 27, 592-596]. According to this analysis the role of Asn(542) in the neprilysin active site is analogous to that of Asn(112 ) of thermolysin, which is to bind the substrate. Site-directed mutage nesis was used to change Asn(542) to Gly or Gin residues; The effect o f these mutations on substrate catalysis and inhibitor binding was exa mined with a series of thiorphan-like compounds containing various deg rees of methylation at the P-2' residue. For both mutated enzymes, det ermination of kinetic parameters with [D-Ala(2),Leu(5)]enkephalin as s ubstrate showed that the large decrease in activity was attributable t o an increase in K-m (14-16-fold) whereas k(cat) values were only slig htly affected (2-3-fold decrease). This is in agreement with Asn(542) being involved in substrate binding rather than directly in catalysis. Finally, the IC50 values for thiorphan and substituted thiorphans str ongly suggest that Asn(542) of neprilysin binds the substrate on the a mino side of the P-2' residue by formation of a unique hydrogen bond.