The ultimate tryptophan residue of neprilysin 2 is not involved in protein maturation and enzymatic activity.
Modeling the three-dimensional structure of neprilysin 2 (NEP2) using the crystal structure of neprilysin as template revealed that their active sites share many common features, though slight differences therein cannot completely account for their specific pharmacological profiles. Recent evidence also suggest that residues outside the active site can play crucial functions in the maturation and enzymatic activity of these metalloproteases. To further explore the functions of amino acids in the acquisition and maintenance of the NEP2 structure, site-directed mutagenesis of conserved residues involved in the enzymatic activity of ECE-1 was performed. In particular, the ultimate tryptophan residue of ECE-1 was recently shown to be important in its activation. This residue was thus mutated in the secreted isoform of NEP2, as were proline residues located in its vicinity. Expression of these mutants in AtT20 cells and study of their secretion and catalytic activities shows that while the ultimate tryptophan residue of the NEP2 sequence is not essential to its proper and activity, structural changes in its vicinity can have a severe impact on the maturation processes involved in the activation of NEP2.
Voisin
Stéphanie
S
INSERM U573, Centre Paul Broca, 2ter rue d'Alésia, 75014 Paris, France.
Ouimet
Tanja
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D045744:Cell Line, Tumor; D017353:Gene Deletion; D006868:Hydrolysis; D015151:Immunoblotting; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015260:Neprilysin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry*
D010455:Peptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014364:Tryptophan; D011392:Proline; D015260:Neprilysin; C476216:neprilysin 2, rat