Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.56 (insulin-degrading enzyme)
 737 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The degradation of insulin by the enzyme insulin protease and by isolated hepatocytes results in proteolytic cleavages in both the A and B chains of intact insulin. Previous studies have shown that one of the A chain cleavages is between A13 leucine and A14 tyrosine and that a second cleavage occurs carboxyl to the A14 residue. In the present study we have used insulin specifically iodinated on the A19 tyrosine and examined the A chain cleavages by the enzyme and by hepatocytes. Insulin degradation products were purified by HPLC and sequenced by automated Edman degradation. Only two A chain cleavage sites were identified, one the previously reported A13-A14 and the other between A14 tyrosine and A15 glutamine. These data thus identify the second A chain cleavage site and further support the role of insulin protease in hepatic metabolism of insulin.
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PMID:Identification of A chain cleavage sites in intact insulin produced by insulin protease and isolated hepatocytes. 330 83

Insulin-degrading enzyme (IDE), a nonlysosomal metalloprotease involved in metabolizing internalized insulin, has catalytic properties that have been strongly conserved through evolution. Two major properties distinguish IDE from the prototypic metalloprotease thermolysin. 1) It is inhibited by cysteine protease inhibitors as well as metalloprotease inhibitors; 2) it contains an inversion of the HEXXH active site motif of thermolysin, where the histidines coordinate zinc and the glutamate participates in catalysis. Furthermore, cysteine is adjacent to the glutamate residue (HXCEH) in human, rat, and Drosophila IDE, although it is not conserved in their close homologue, Escherichia coli protease III. This cysteine has been postulated to mediate the differential sensitivity of IDE and protease III to cysteine protease inhibitors and chelators. The role of the cysteine in IDE catalysis and inhibitor sensitivity was examined by mutating Cys110 to glycine or serine. To determine whether glutamate in this unusual motif participates in catalysis, we mutated Glu111 to aspartate, valine, or glutamine. Vectors containing wild type or mutant enzymes were transfected into COS cells, and expression was confirmed by Western blotting. Although the glutamate mutants were devoid of insulin degrading activity, the cysteine mutants were indistinguishable from wild type enzyme in both catalytic activity and sensitivity to inhibitors. The loss of activity in the glutamate mutants was not due to gross alterations in tertiary structure, as shown by retention of the ability to bind substrate and by conservative and nonconservative mutation of a neighboring residue with no apparent effect on catalysis. These results demonstrate that the conserved glutamate in the zinc-binding site of human insulin-degrading enzyme is a major catalytic residue, while a conserved cysteine in this region is not essential for catalysis or inhibitor sensitivity.
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PMID:Functional analysis of conserved residues in the active site of insulin-degrading enzyme. 810 41

Insulin-degrading enzyme is a nonlysosomal metalloprotease that initiates degradation of internalized insulin in some cells. We previously identified a potential catalytic site containing an inversion of the Zn(2+)-binding domain of the thermolysin family (Kuo, W.-L., Gehm, B. D., and Rosner, M. R. (1991) Mol. Endocrinol. 4, 1580-1591). The role of this site in catalysis was examined by mutating one of the presumptive Zn(2+)-coordinating histidines (His108) in human insulin-degrading enzyme to leucine or glutamine, which were predicted to reduce or eliminate Zn2+ binding without substantially altering secondary structure. cDNAs for the mutant and wild-type enzymes were incorporated into an expression vector and transfected into COS cells. Expression of the transfected genes was confirmed by Northern and Western blots. In contrast to the wild-type gene, which increased insulin degradation by cell extracts and intact cells several-fold, the mutated genes had no effect on insulin degradation, indicating a loss of catalytic activity. However, the mutants' ability to bind substrate was unimpaired, as affinity labeling with 125I-insulin was increased compared to the wild type. These results suggest that an intact Zn(2+)-binding domain in human insulin-degrading enzyme is required for catalytic activity and can affect, but is not required for, substrate binding.
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PMID:Mutations in a zinc-binding domain of human insulin-degrading enzyme eliminate catalytic activity but not insulin binding. 846 15