1 Mixed Dimers of Insulin

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 beta 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. Insulysin (insulin degrading enzyme, IDE, EC 3.4.14.56) is one of the major enzymes involved in Aβ clearance as evidenced by genetic studies with IDE deficient mice (1) as well as over by expression of IDE in transgenic mouse brain (2). IDE is also the major enzyme involved in cellular insulin degradation (3-5) and mutations in IDE in the GK rat have been linked to type 2 diabetes (6). IDE is made up of four structurally similar domains arranged into a U-like conformation, with only the N terminal most domain retaining a functional active site (9). The enzyme is primarily dimeric (7-10) and unique among the zinc metalloproteases in that it exhibits non-classical kinetics with small synthetic peptide substrates (8). This property appears dependent on association to form a homodimer (8). With small synthetic peptide substrates IDE exhibits a sigmoidal rate versus substrate response indicative of allosteric enzymes showing homotropic activation or activation by substrate. In addition IDE is activated by small physiological peptides such as bradykinin and dynorphin (8) by what can be termed “heterotrophic activation” or activation by molecules other than the substrate. We recently obtained evidence that peptide activators bind to a site on the enzyme we refer to as the distal site (Noinij et al., submitted). Both this distal site as well as the active site was found to contain a peptide derived from TEV protease release of the polyHis and linker region from an inactive IDE fusion protein. As might be expected with active IDE neither the active site nor the distal site is occupied by the same TEV protease treatment, presumably due to degradation of the released polyHis containing peptide. This distal site makes extended binding http://www.jbc.org/cgi/doi/10.1074/jbc.M110.191668 The latest version is at JBC Papers in Press. Published on February 22, 2011 as Manuscript M110.191668