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.
Highlights
Insulysin is one of the major enzymes involved in A clearance as evidenced by genetic studies with inactive subunit can bind substrate (IDE)-deficient mice (1) as well as by over expression of IDE in transgenic mouse brain (2)
We have found that mutation of a tyrosine within this distal binding site, Tyr-609, disrupts allosteric activation, and we have proposed that this distal site is an allosteric binding site
Because IDE functions primarily as a dimer we set out to determine whether binding at the substrate binding site and binding at the distal binding site can produce allosteric activation by a cis or trans mechanism
Summary
Preparation of Mixed Oligomers of IDE—Mixed oligomers, of IDE were generated by using baculovirus to co-express wild type and IDE mutant forms in Sf9 insect cells (8). Oligomeric forms were generated in 200-ml cultures expressed for 72 h and isolated by Ni-affinity chromatography using a 2-ml HIS-select Ni-NTA-agarose column as previously described (16, 17) This method yielded catalytically active oligomers composed of the His-tagged inactive subunit and non-tagged catalytically active subunits. Reactions (200 l) containing 0.5 M A1–40-Lys(Biotin)FAM-labeled in 50 mM Tris-HCl buffer, pH 7.4, plus 0.05% Tween-20 were incubated with 50 – 400 ng of IDE for 30 min. Amyloid Peptide Binding to IDE—The binding of the fluorescent A1–40 analog A1–40 FAM-labeled (AnaSpec, Inc.) to IDE was measured by following the change in fluorescence anisotropy when increasing amounts of IDE (0 –3 M) was added to 1 M FAM-labeled A1–40 in 50 mM Tris-HCl, pH 7.4 buffer in a total volume of 200 l.
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