MUTAGENESIS OF GLU(403) TO CYS IN RABBIT NEUTRAL ENDOPEPTIDASE-24.11 (NEPRILYSIN) CREATES A DISULFIDE-LINKED HOMODIMER - ANALOGY WITH ENDOTHELIN-CONVERTING ENZYME

Neutral endopeptidase-24.11 (NEP; neprilysin; EC 3.4.24.11) and endoth elin-converting enzyme (ECE) are related zinc metallopeptidases involv ed in the processing of biologically active peptides. Only ECE, howeve r, exists as a disulphide-linked homodimer. The covalent linkage in ra t ECE is between Cys(412) in each subunit, which is equivalent to Glu( 403) in rabbit NEP. Here we report that directed mutagenesis of Glu(40 3) to cysteine in rabbit NEP creates a disulphide-linked homodimer, as revealed by transient transfection in COS-1 cells and SDS/PAGE of a m embrane fraction. Under reducing conditions, both the mutant (E403C) a nd the wild-type NEP migrate as a polypeptide of 92 kDa. However, unde r non-reducing conditions, the M-r of the wild type remains unchanged, whereas that of the mutant is doubled. Co-transfection of wild-type E CE and E403C NEP cDNA did not result in the production of a NEP-ECE he terodimer. Comparison of the kinetic constants for wild-type and E403C mutant NEP with either [D-Ala(2),Leu(5)]enkephalin or boxypropanoyl-a lanyl-alanyl-leucine-4-nitroanilide (Suc-Ala-Ala-Leu-NH-Np) as substra te show a decrease of approx. 50% in V-max/K-m for the mutant form. Th e IC50 value for inhibition of the mutant by phosphoramidon or thiorph an is increased 3-fold and 5-fold respectively. Although NEP and ECE e xhibit only about 40 % identity and differ substantially in substrate specificity and some other characteristics, these data indicate that t hey have considerable similarity in three-dimensional structure, allow ing dimer formation in the mutant NEP with the disulphide link probabl y occurring in a hydrophilic surface loop.