Mutation of Active Site Residues of Insulin-degrading Enzyme Alters Allosteric Interactions

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

doi:10.1074/jbc.m501896200

Louis B. Hersh, Maria Aparecida Juliano + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m501896200

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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-GGFLRKHGQ-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 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.

Highlights

It is generally accepted that the accumulation of amyloid ␤-peptides (A␤),1 amyloid ␤-peptide 1– 42, plays a central role in the pathogenesis of Alzheimer disease (AD)
In order to determine whether the mutations introduced into insulin-degrading enzyme (IDE) affected enzyme specificity, we looked at the cleavage pattern of three physiological peptides, ␤-endorphin, insulin, and amyloid ␤ peptide 1– 40 using each of the mutants listed in Effect of ATP and PPPi on mutant IDE activity
Mutation of residues within the active site motif (HEXXH or HXXEH) of zinc metallopeptidases has been reported to lead to a loss of catalytic activity

Summary

Introduction

It is generally accepted that the accumulation of amyloid ␤-peptides (A␤), amyloid ␤-peptide 1– 42, plays a central role in the pathogenesis of Alzheimer disease (AD). There are recent reports (6 – 8) that the ␤-secretase involved in amyloid ␤-peptide synthesis is increased in sporadic AD patients. Whether this increase is a cause of or a consequence of AD and whether this increase in activity will be seen in larger patient populations remains to be established. A linkage of a region of chromosome 10q to late onset Alzheimer disease has been reported by several groups [22,23,24,25]. This region of the chromosome is within 195 kilobases of the IDE gene. We have recently shown that IDE exhibits allosteric properties being

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