Identification of glutamate-169 as the third zinc-binding residue in proteinase III, a member of the family of insulin-degrading enzymes.
A novel active site has been identified in a family of zinc-dependent metalloendopeptidases that includes bacterial proteinase III, the human and Drosophila insulin-degrading enzymes, and the processing-enhancing protein subunit of the mitochondrial processing proteinase. None of these enzymes contains the conserved active site described in most other metalloendopeptidases, HEXXH; instead, all four contain an inversion of this motif, HXXEH. Prior mutagenesis studies of proteinase III indicate that the two histidines are essential for co-ordinating the zinc atom, while all three residues are required for enzyme activity. To identify the third zinc-binding residue in this protein family, three glutamates downstream from the active site were mutated to glutamine in proteinase III. The mutant proteins were expressed and their ability to degrade insulin was compared with the wild-type enzyme. The glutamate-204 mutant was as active as the wild-type protein, the glutamate-162 mutant retained 20% of the activity of the wild-type enzyme and the glutamate-169 mutant was completely devoid of insulin-degrading activity. The purified wild-type and glutamate-204 mutant enzymes were found to contain nearly stoichiometric levels of zinc by atomic absorption spectrophotometry, whereas the glutamate-162 mutant had a slight reduction in the level of zinc, and the glutamate-169 mutant retained less than 0.3 mol of zinc/mol of enzyme. These findings are consistent with glutamate-169 being the third zinc-binding residue in proteinase III.
Becker
A B
AB
Department of Pharmacology, Stanford University School of Medicine, CA 94305.
Roth
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004330:Drosophila; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D005971:Glutamates / Q000032:analysis*; D018698:Glutamic Acid; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D015032:Zinc / Q000378:metabolism*
D005971:Glutamates; D007328:Insulin; D018698:Glutamic Acid; D010450:Endopeptidases; D008666:Metalloendopeptidases; C046583:pitrilysin; D015032:Zinc