ASP650 IS CRUCIAL FOR CATALYTIC ACTIVITY OF NEUTRAL ENDOPEPTIDASE-24-11

Neutral endopeptidase (NEP) is a membrane-bound mammalian ectopeptidas e that contains a catalytic zinc ion in its active site. Previous stud ies showed that the active site, and especially the zinc-binding site of NEP, have features in common with the prototypical bacterial zinc p rotease, thermolysin. Sequence comparison reveals that both enzymes ha ve a conserved Asp residue (Asp650 in NEP and Asp170 in thermolysin) l ocated four positions on the C-side of the third zinc ligand. In therm olysin, this residue is involved in a carboxylate-histidine-zinc inter action whose functional role has never been established [Christianson, D. W. and Alexander, R. S. (1990) Nature 346, 225]. To test the hypot hesis that, in NEP, this residue is important for catalysis, we have c hanged Asp650 of NEP by site-directed mutagenesis and expressed the mu tant enzymes in COS-1 cells. Substitution of Glu, Asn or Ala for Asp65 0 resulted in mutant enzymes exhibiting drastic decreases in specific activity. Binding experiments using the zinc-chelating inhibitor roxya mino)-1,4-dioxo-2-(phenylmethyl)butyl]glycine suggested that the zinc ion is present in the active site of these mutant enzymes. These resul ts strongly support the conclusion that Asp650 in NEP is crucial for h ydrolytic activity.