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Size and Stability are Critical Elements in the Substrate Selectivity of Insulin‐Degrading Enzyme
Author(s) -
Kalas Vasilios,
Ralat Luis A,
Zheng Zhongzhou,
Sosnick Tobin R,
Tang WeiJen
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.463.8
Subject(s) - chemistry , substrate (aquarium) , selectivity , proteases , peptide , protease , hydrolase , enzyme , proteolysis , cleavage (geology) , stereochemistry , insulin degrading enzyme , biochemistry , biology , ecology , paleontology , fracture (geology) , catalysis
Insulin‐degrading enzyme (IDE) is a zinc metalloprotease involved in the clearance of small bioactive peptides, such as insulin and amyloid‐beta. Given the considerable diversity in substrate tertiary structure, the criteria for substrate recognition and degradation by this protease have been difficult to determine. Here, we evaluate size and local stability as parameters in the substrate selectivity of IDE using opioid peptides and ubiquitin variants as model systems, respectively. Kinetic analyses of IDE, using opioid peptides ranging from 5 to 31 residues, demonstrate that opioid peptides containing 17 or more residues exhibit K m values of 50 μM or less and k cat values that do not exceed 40 s −1 . In contrast, smaller opioid peptides exhibited K m values above 900 μM and k cat values above 600 s −1 . Mutations at conserved substrate binding sites in IDE are currently being investigated to identify the molecular basis of substrate size discrimination. To probe local stability as a parameter in substrate selectivity, we mutated residues on each of the seven major structural elements of ubiquitin (Ub), a 76‐residue inhibitor of IDE, which destabilize Ub by 0.5–9 kcal/mol. Surprisingly, Ub variants which lose at least 1.6 kcal/mol in local stability are cleaved by IDE with k cat values above 0.5 sec −1 . While the first cut site is identical in all digested variants, the cleavage pattern varies with increasing instability of Ub proteins. Together, these findings imply that changing a substrate's property modifies the enzyme's specificity and thus suggest a knowledge basis for the rational design of peptidomimetic modulators of IDE activity.