Mutation of active site residues of insulin-degrading enzyme alters allosteric interactions

Eun Suk Song, Abigail Daily, Michael G. Fried, Maria Aparecida Juliano, Luiz Juliano, Louis B. Hersh

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33 Scopus citations


The active site glutamate (Glu111) and the active site histidine (His112) of insulin-degrading enzyme (IDE) were mutated. These mutant enzymes exhibit, in addition to a large decrease in catalytic activity, a change in the substrate-velocity response from a sigmoidal one seen with the native enzyme (Hill coefficient > 2), to a hyperbolic response. With 2-aminobenzoyl-GGFLKKH-GQ-N-(2,4-dinitrophenyl)ethylenediamine as substrate, ATP and triphosphate increase the reaction rate of the wild type enzyme some 50-80-fold. This effect is dampened with glutamate mutants to no effect or less than a 3-fold increase in activity and changed to inhibition with the histidine mutants. Sedimentation equilibrium shows the IDE mutants exhibit a similar oligomeric distribution as the wild type enzyme, being predominantly monomeric, with triphosphate having little if any effect on the oligomeric state. Triphosphate did induce aggregation of many of the IDE mutants. Thus, the oligomeric state of IDE does not correlate with kinetic properties. The His 112 mutants were shown to bind zinc, but with a lower affinity than the wild type enzyme. The glutamate mutants displayed an altered cleavage profile for the peptide β-endorphin. Wild type IDE cleaved β-endorphin at Leu17-Phe18 and Phe18-Lys19, whereas the glutamate mutants cleaved at these sites, but in addition at Lys19-Asn20 and at Met5-Thr6. Thus, active site mutations of IDE are suggested to not only reduce catalytic activity but also cause local conformational changes that affect the allosteric properties of the enzyme.

Original languageEnglish
Pages (from-to)17701-17706
Number of pages6
JournalJournal of Biological Chemistry
Issue number18
StatePublished - May 6 2005

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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