From 2007.igem.org

insulin-degrading enzyme [Homo sapiens]. [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&id=55959215 1019 aa]     [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=184555 mRNA 3337 bp]
[http://www.expasy.org/enzyme/3.4.24.56 EC 3.4.24.56]
[http://www.expasy.org/uniprot/P14735 UniProt] 

• (Wikipedia)
  • Known alternatively as insulysin or insulin protease
  • it has since been shown to have additional substrates, including the signaling peptides, glucagon, TGF alpha, and β-endorphin
  • IDE can degrade amyloid beta (Aβ), a peptide implicated in the pathogenesis of Alzheimer’s disease
  • IDE can exist in two conformations: an open conformation, in which substrates can access the active site, and a closed state, in which the active site is contained within the chamber formed by the two concave domains. Targeted mutations that prevent the closed conformation result in a 40-fold increase in catalytic activity
• Camberos, M.C., Perez, A.A., Udrisar, D.P., Wanderley, M.I., Cresto, J.C. ATP Inhibits Insulin-Degrading Enzyme Activity Experimental Biology and Medicine 2001 226: 334-341
  • One to 5 mM ATP induced a dose-dependent inhibition of insulin degradation.
    File:ATP inhibition of insulin degradation.GIF
  • Kinetic analysis of ATP inhibition suggested an allosteric effect as the plot of 1/v (insulin degradation) versus ATP concentration was not linear and the Hill coefficient was more than 1(1.51 and 2.44).
  • The binding constant for allosteric inhibition was K_i^T = 1.5 × 10−7 M showing a decrease of enzyme affinity induced by ATP.
  • It has been demonstrated that 5 mM extracellular ATP inhibits the insulin degradation in isolated rat adipocytes, suggesting that extracellular ATP may be a signal for inhibition in the degradative pathway of insulin processing.
  • Inhibition of insulin degradation was found in rat cytosol treated with aluminum fluoride or phosphatidylserine plus ATP
  • Addition of 10 mM MgCl2 to the incubation media increases insulin degradation
  • Zn²⁺ chelation denatures IDE, which lost its activity, and Zn²⁺ and Mn²⁺ ions protect IDE from denaturation. Mn²⁺ and Mg²⁺ ions are required for ATP inhibition, and the effect of 3 mM ATP on insulin degradation is half in the absence of these ions in the incubation buffer
  • The double reciprocal plot of insulin degradation by IDE gives a straight line with an apparent Km of 58 × 10⁻⁹ M.
  • Insulin degradation by IDE shows a decrease in V_max with increasing doses of ATP (V_max, M/hr; IDE =1.32 × 10⁻⁸; IDE + 0.3 mM ATP=0.95 × 10⁻⁸; IDE + 3 mM ATP =0.49 × 10⁻⁸)
  • There is a significant decrease in the Vmax of insulin degradation between control and IDE plus 3 mM ATP (P < 0.05) with a similar Km (IDE + 0.3 mM ATP, Ki: 56 × 10⁻⁹ M; IDE + 3 mM ATP, Ki: 45 × 10⁻⁹ M; see inset).
  • Nevertheless, cellular processing of insulin and glucagon are different. Insulin degradation is an intracellular event, and glucagon degradation is almost all extracellular.
  • ATP inhibits the insulin degradation by IDE. It is also known that the enzyme is found in the cytosol, the same as insulin after its internalization.
• María del Carmen Camberos, Maria, Cresto, Juan C. Insulin-Degrading Enzyme Hydrolyzes ATP Experimental Biology and Medicine 2007 232: 281-292
  • IDEs have ATPase activity and that insulin-binding and degradation are dependent on ATP concentration.
  • IDE degrades not only insulin and glucagon but also insulin-like growth factors I and II, the leader peptide of peroxysomal thiolase.
  • Cleavage site studies pointed out that substrate recognition by IDE would more likely depend on the tertiary peptide conformation rather than on the amino acid sequence.
  • A common motif in the sequences of IDE substrates, HNHHHPSH, where H is wholly or partly hydrophobic character, N is small and neutral, P is polar, and S is polar and/or small amino acid residue.

• Yuequan Shen et al. Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism. Nature 443, 7113 (19 Oct 2006) 
  Arrows indicate the main cleavage sites of the substrate by IDE

  File:Cleavage sites of IDE.GIF

   

• [http://www.boomer.org/pkin/PK06/PK2006274.html http://www.boomer.org/pkin/PK06/PK2006274.html]
  Due to insulin degradation enzyme (IDE) released mainly from red blood cells, we have seen the recoveries of both endogenous and exogenous insulin were significantly decreased in serum after freeze/thaw and bench top as well as in hemolyzed serum or plasma samples. EDTA as a protease inhibitor (PI) plus Bacitracin as a PI too, significantly increased stability of insulin in normal and hemolysed plasma. BAC is a strong IDE inhibitor that can be used for preventing insulin degradation during sample preparation, storage and analysis.

• [http://www.ebmonline.org/cgi/content/full/226/4/334 ATP Inhibits Insulin-Degrading Enzyme Activity. Experimental Biology and Medicine 226:334-341 (2001).]

  Insulin-degrading enzyme (IDE) is an evolutionarily conserved protein. In vitro, IDE not only degrades insulin and glucagon, but also insulin growth factors I and II, the leader peptide of peroxysomal prethiolase transforming growth factor, the ß-amyloid peptide, and other peptides. In vivo, IDE degrades insulin and transforming growth factor. Cleavage sites studies suggest that substrate recognition by IDE would depend on the tertiary peptide conformation more than on an amino acid sequence. Degradation activity by IDE is abolished by the Zn²⁺ chelator 1,10-phenantroline, the insulin-binding inhibitor bacitracin, the thiol blocking agents p-hydroxy-mercuribenzoate, and N-ethylmaleimide among others.

• [http://en.wikipedia.org/wiki/Bacitracin Bacitracin]

  - Bacitracin is a mixture of related cyclic polypeptides produced by organisms of the licheniformis group of Bacillus subtilis var Tracy. Its unique name derives from the fact that the bacillus producing it was first isolated in 1943 from a knee scrape from a girl named Margaret Tracy. As a toxic and difficult-to-use antibiotic, bacitracin doesn't work well orally. However, it is very effective topically.

  - Bacitracin is synthesised via the so-called nonribosomal peptide synthetases (NRPSs), which means that ribosomes are not involved in its synthesis.

  - As bacitracin zinc salt, and in combination with other topical antibiotics (usually polymyxin B and neomycin), it is used in ointment form for topical treatment of a variety of localized skin and eye infections, as well as for the prevention of wound infections. In the United States a popular brand name Neosporin contains Bacitracin as one of its antibiotic agents along with Neomycin and Polymyxin B.