Substrate Activation of Insulin-degrading Enzyme (Insulysin)

Abstract

The rate of the insulin-degrading enzyme (IDE)-catalyzed hydrolysis of the fluorogenic substrate 2-aminobenzoyl-GGFLRKHGQ-ethylenediamine-2,4-dinitrophenyl is increased 2-7-fold by other peptide substrates but not by peptide non-substrates. This increased rate is attributed to a decrease in K m with little effect on V max . An ϳ2.5-fold increase in the rate of amyloid ␤ peptide hydrolysis is produced by dynorphin B-9. However, with insulin as substrate, dynorphin B-9 is inhibitory. Immunoprecipitation of differentially tagged IDE and gel filtration analysis were used to show that IDE exists as a mixture of dimers and tetramers. The equilibrium between dimer and tetramer is concentration-dependent, with the dimer the more active form. Bradykinin shifted the equilibrium toward dimer. Activation of substrate hydrolysis is not seen with a mixed dimer of IDE containing one active subunit and one subunit that is catalytically inactive and deficient in substrate binding. On the other hand, a mixed dimer containing one active subunit and one subunit that is catalytically inactive but binds substrate with normal affinity is activated by peptides. These findings suggest that peptides bind to one subunit of IDE and induce a conformational change that shifts the equilibrium to the more active dimer as well as activates the adjacent subunit. The selective activation of IDE toward amyloid ␤ peptide relative to insulin suggests the potential for development of compounds that increase IDE activity toward amyloid ␤ peptide as a therapeutic intervention for the treatment of Alzheimer's disease.

Glucose inhibits the insulin-induced activation of the insulin-degrading enzyme in HepG2 cells

“…However, we detected no regulation of IDE mRNA and protein levels by insulin at normal glucose concentration. The binding of some peptide substrates to one of the IDE subunits is known to lead to allosteric regulation of IDE activity, with the induction of a shift of the IDE dimer/ tetramer equilibrium to the more active dimer and activation of the adjacent subunit [29]. However, insulin binding was shown to induce no activation of IDE, probably because insulin, which is a dimer of A and B chains, can simultaneously bind to both subunits of the IDE dimer.…”

Anion Activation Site of Insulin-degrading Enzyme

“…Crystallization and data collection conditions were the same as used for the ATPbound structure except ATP and EDTA were not included in the final soaking solution. activation (20). Hill coefficients of 1.7 were calculated for both mutants, which is slightly less than the value of 2.1 obtained for IDE.…”

“…IDE is unusual among zinc metallopeptidases in that it exhibits allosteric kinetic behavior, with small peptide substrates increasing the activity of the enzyme toward the same or other small peptides (20). In addition, IDE has been shown to be activated heterotropically by polyphosphate anions (21,22).…”

“…IDE exists as an equilibrium mixture of monomers, dimers, and tetramers, with dimers thought to be the predominant and most active state (11,20). IDE is unusual among zinc metallopeptidases in that it exhibits allosteric kinetic behavior, with small peptide substrates increasing the activity of the enzyme toward the same or other small peptides (20).…”

“…In the studies presented here, we identify the site where ATP (and likely other anions) binds to IDE using x-ray crystallography and show that mutating enzyme residues in this site sharply decreases ATP/anion activation. IDE (residues 41-1019), bearing the mutations K898A,K899A,S901A (IDE K898A,K899A,S901A ), or R429S (IDE R429S ) were prepared from the pFastBac HTb-IDE plasmid (20,25) using the QuikChange mutagenesis kit (Stratagene). Primers used for mutagenesis are listed as follows with the base changes in boldface and underlined: 1) Lys-898, Lys-899, and Ser-901 changed to alanine-IDE K898A,K899A,S901A : K898A forward, 5Ј-CGACTCGACAAACCAGCGAAACTC-TCTGCAGAG-3Ј, and reverse, 5Ј-CTCTGCAGAGAGTTT-CGCTGGTTTGTCGAGTCG-3Ј; K899A forward, 5Ј-CTC-GACAAACCAGCGGCACTCTCTGCAGAGTGC-3Ј, and reverse, 5Ј-GCACTCTGCAGAGAGTGCCGCTGGTTTGT-CGAG-3Ј; S901A forward, 5Ј-AAACCAGCGGCACTCGCT-GCAGAGTGCGCGAAG-3Ј, and reverse, 5Ј-CTTCGCGC-ACTCTGCAGCGAGTGCCGCTGGTTT-3Ј; 2) Arg-429 converted to serine IDE R429S , R429S forward, 5Ј-TTTAAAG-ATAAAGAGAGCCCACGAGGCTACACA-3Ј, and reverse, 5Ј-TGTGTAGCCTCGTGGGCTCTCTTTATCTTTAAA-3Ј.…”