title: Mixed dimers of insulin-degrading enzyme reveal a cis activation mechanism.
abstract: Insulin-degrading enzyme (IDE) exists primarily as a dimer being unique among the zinc metalloproteases in that it exhibits allosteric kinetics with small synthetic peptide substrates. In addition the IDE reaction rate is increased by small peptides that bind to a distal site within the substrate binding site. We have generated mixed dimers of IDE in which one or both subunits contain mutations that affect activity. The mutation Y609F in the distal part of the substrate binding site of the active subunit blocks allosteric activation regardless of the activity of the other subunit. This effect shows that substrate or small peptide activation occurs through a cis effect. A mixed dimer composed of one wild-type subunit and the other subunit containing a mutation that neither permits substrate binding nor catalysis (H112Q) exhibits the same turnover number per active subunit as wild-type IDE. In contrast, a mixed dimer in which one subunit contains the wild-type sequence and the other contains a mutation that permits substrate binding, but not catalysis (E111F), exhibits a decrease in turnover number. This indicates a negative trans effect of substrate binding at the active site. On the other hand, activation in trans is observed with extended substrates that occupy both the active and distal sites. Comparison of the binding of an amyloid β peptide analog to wild-type IDE and to the Y609F mutant showed no difference in affinity, indicating that Y609 does not play a significant role in substrate binding at the distal site.
authors.affiliation: Department of Molecular and Cellular Biochemistry and the Center for Structural Biology, University of Kentucky, Lexington, Kentucky 40536, USA.
authors.forename: Eun Suk
authors.initials: ES
authors.lastname: Song
authors.forename: David W
authors.initials: DW
authors.lastname: Rodgers
authors.forename: Louis B
authors.initials: LB
authors.lastname: Hersh
mesh_terms: D016229:Amyloid beta-Peptides / Q000737:chemistry; D000818:Animals; D001920:Bradykinin / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D019281:Dimerization; D004399:Dynorphins / Q000737:chemistry; D004723:Endorphins / Q000737:chemistry; D006801:Humans; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D016229:Amyloid beta-Peptides; D004723:Endorphins; D007328:Insulin; D010455:Peptides; D004399:Dynorphins; C036200:rimorphin; D001920:Bradykinin