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 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 Rogues (1988) 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 mutagenesis was used to change Asn(542) to Gly or Gin 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 P-2' residue. For both mutated enzymes, determination of kinetic parameters with [D-Ala(2),Leu(5)]enkephalin as substrate showed that the large decrease in activity was attributable to an increase in K-m (14-16-fold) whereas k(cat) values were only slightly 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 strongly suggest that Asn(542) of neprilysin binds the substrate on the amino side of the P-2' residue by formation of a unique hydrogen bond.