Identification of glutamic acid 646 as a zinc-coordinating residue in endopeptidase-24.11

Neutral endopeptidase (EC 3.424.11, NEP) is a membrane-bound zinc-metallopeptidase. The substrate specificity and catalytic activity of NEP resemble those of thermolysin, a bacterial zinc-metalloprotease. Comparison of the primary structure of both enzymes suggests that several amino acids present in the active site of thermolysin are also found in NEP. Using site-directed mutagenesis of the cDNA encoding the NEP sequence, we have already shown that His residues 583 and 587 are two of the three zinc ligands. In order to identify the third zinc ligand, we have substituted Val or Asp for Glu616 or Glu646. Val616 NEP showed the same kinetic parameters as the non-mutated NEP. In contrast, the mutant Val646 NEP was almost completely devoid of catalytic activity and unable to bind the tritiated inhibitor [3H]N-[2(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxypropyl]gl ycine, the binding of which is dependent on the presence of the zinc ion. Replacing Glu for Asp at position 646 conserved the negative charge, and the mutant enzyme exhibited the same Km value as the non-mutated enzyme, but kCat was decreased to less than 3% of the value of the non-mutated enzyme. When compared to the non-mutated enzyme Asp646 NEP showed a higher susceptibility to chelating agents, but bound the tritiated inhibitor with the same affinity. Taken together, these observations strongly suggest that Glu646 of NEP is the third zinc-coordinating residue and is equivalent to Glu166 in thermolysin.