Asp650 is crucial for catalytic activity of neutral endopeptidase 24-11.
Neutral endopeptidase (NEP) is a membrane-bound mammalian ectopeptidase that contains a catalytic zinc ion in its active site. Previous studies showed that the active site, and especially the zinc-binding site of NEP, have features in common with the prototypical bacterial zinc protease, thermolysin. Sequence comparison reveals that both enzymes have a conserved Asp residue (Asp650 in NEP and Asp170 in thermolysin) located four positions on the C-side of the third zinc ligand. In thermolysin, this residue is involved in a carboxylate-histidine-zinc interaction whose functional role has never been established [Christianson, D. W. & Alexander, R. S. (1990) Nature 346, 225]. To test the hypothesis that, in NEP, this residue is important for catalysis, we have changed Asp650 of NEP by site-directed mutagenesis and expressed the mutant enzymes in COS-1 cells. Substitution of Glu, Asn or Ala for Asp650 resulted in mutant enzymes exhibiting drastic decreases in specific activity. Binding experiments using the zinc-chelating inhibitor [3H]-N-[(2RS)-4-(hydroxyamino)-1,4-dioxo-2-(phenylmethyl)butyl]glycine suggested that the zinc ion is present in the active site of these mutant enzymes. These results strongly support the conclusion that Asp650 in NEP is crucial for hydrolytic activity.
Le Moual
H
H
Département de Biochimie, Université de Montréal, Canada.
Dion
N
N
Roques
B P
BP
Crine
P
P
Boileau
G
G
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D015870:Gene Expression; D006868:Hydrolysis; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D015260:Neprilysin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013820:Thermolysin / Q000737:chemistry; D014162:Transfection; D015032:Zinc / Q000378:metabolism
D001224:Aspartic Acid; D015260:Neprilysin; D013820:Thermolysin; D015032:Zinc