Mutagenesis of Glu super(403) to Cys in rabbit neutral endopeptidase-24.11 (neprilysin) creates a disulphide-linked homodimer: Analogy with endothelin-converting enzyme

• Published in: Biochemical journal Vol. 327; no. 3; pp. 925 - 929
• Main Authors: Hoang, Mien V, Sansom, CE, Turner, A J
• Format: Journal Article
• Language: English
• Published: 01.11.1997
• ISSN: 0264-6021

Neutral endopeptidase-24.11 (NEP; neprilysin; EC 3.4.24.11) and endothelin-converting enzyme (ECE) are related zinc metallopeptidases involved in the processing of biologically active peptides. Only ECE, however, exists as a disulphide-linked homodimer. The covalent linkage in rat ECE is between Cys super(412) in each subunit, which is equivalent to Glu super(403) in rabbit NEP. Here we report that directed mutagenesis of Glu super(403) to cysteine in rabbit NEP creates a disulphide-linked homodimer, as revealed by transient transfection in COS-1 cells and SDS/PAGE of a membrane fraction. Under reducing conditions, both the mutant (E403C) and the wild-type NEP migrate as a polypeptide of 92 kDa. However, under non-reducing conditions, the M sub(r) of the wild type remains unchanged, whereas that of the mutant is doubled. Co-transfection of wild-type ECE and E403C NEP cDNA did not result in the production of a NEP-ECE heterodimer. Comparison of the kinetic constants for wild-type and E403C mutant NEP with either [D-Ala super(2),Leu super(5)]enkephalin or 3-carboxypropanoyl-alanyl-alanyl-leucine-4-nitroanilide (Suc-Ala-Ala-Leu-NH-Np) as substrate show a decrease of approx. 50% in V sub(max)/K sub(m) for the mutant form. The IC sub(50) value for inhibition of the mutant by phosphoramidon or thiorphan is increased 3-fold and 5-fold respectively. Although NEP and ECE exhibit only about 40% identity and differ substantially in substrate specificity and some other characteristics, these data indicate that they have considerable similarity in three-dimensional structure, allowing dimer formation in the mutant NEP with the disulphide link probably occurring in a hydrophilic surface loop.