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

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