IDENTIFICATION OF ZINC LIGANDS OF THE INSULIN-DEGRADING ENZYME

The insulin degrading enzyme (IDE), a nonlysosomal enzyme involved in the metabolism of internalized insulin, is a member of a new family of metalloproteases which has an HXXEH active site motif. We have previously shown that both His(108) and Glu(111) within the HX-CEH domain of human IDE are necessary for catalytic activity. Comparison to the prototypic zinc metalloprotease thermolysin, which contains an inversion of this motif, would predict that His(112), as well as a downstream glutamate, serves as the second and third zinc Ligands of IDE. To examine the role of His(112), we mutated this residue to glutamine, leucine, or arginine. To identify a downstream zinc ligand, we substituted a glutamine for glutamate at either Glu(182) or Glu(189), both of which are conserved in human, rat, and Drosophila IDE. Vectors containing wild type or mutant IDE genes were transfected into COS cells, and the enzymes were analyzed for insulin degradation, insulin cross-linking, and zinc binding. Our results suggest that His(108), His(112), and Glu(189) are the zinc ligands of human IDE, and Glu(182) can influence zinc binding. In addition to a catalytic role, zinc binding to these residues appears to play a role in stabilizing the structure of the enzyme.