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

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

Author Song, E. S. Google Scholar
Daily, A. Google Scholar
Fried, M. G. Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Hersh, L. B. Google Scholar
Institution Univ Kentucky
Universidade Federal de São Paulo (UNIFESP)
Abstract The active site glutamate (Glu(111)) and the active site histidine (His(112)) 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-GGFLRKHGQN-(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 beta-endorphin. Wild type IDE cleaved beta-endorphin at Leu(17)-Phe(18) and Phe(18)-Lys(19), whereas the glutamate mutants cleaved at these sites, but in addition at Lys(19)-Asn(20) and at Met(5)-Thr(6). 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.
Language English
Date 2005-05-06
Published in Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 280, n. 18, p. 17701-17706, 2005.
ISSN 0021-9258 (Sherpa/Romeo, impact factor)
Publisher Amer Soc Biochemistry Molecular Biology Inc
Extent 17701-17706
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000228807200019

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