Analysis of the Subsite Specificity of Rat Insulysin Using Fluorogenic Peptide Substrates

Abstract

Recombinant rat insulysin was shown to cleave the internally quenched fluorogenic peptide 2-aminobenzyl-GGFLRKVGQ-ethylenediamine-2,4-dinitrophenol at the R-K bond, exhibiting a K m of 13 M and a V max of 2.6 mol min ؊1 mg ؊1 . Derivatives of this peptide in which the P 2 leucine or the P 2 ' valine were replaced with other residues were used to probe the subsite specificity of the enzyme. Varying the P 2 residue produced a 4-fold range in K m and a 7-fold range in k cat . The nature of the P 2 residue had a significant effect on the site of cleavage. Leucine, isoleucine, valine, and aspartate produced cleavage at the R-K bond. Asparagine produced 36% cleavage at the N-R bond and 64% cleavage at the R-K bond, whereas with alanine or serine the A-R and S-R bonds were the major cleavage sites. With tyrosine, phenylalanine, methionine, or histidine representing the varied residue X, cleavages at F-X, X-R, and R-K were seen, whereas with tryptophan equal cleavage occurred at the F-W and W-R bonds. Variable P 2 ' residues produce less of a change in both K m and k cat and have little influence on the cleavage site. Exceptions are phenylalanine, tyrosine, leucine, and isoleucine, which in addition to producing cleavage at the R-K bond, produce significant cleavage at the L-R bond. Alanine and tyrosine were unique in producing cleavage at the F-L bond. Taken together, these data suggest that insulysin specificity is directed toward the amino side of hydrophobic and basic residues and that the enzyme has an extended substrate binding site.Insulysin (insulin-degrading enzyme; EC 3.4.24.56) was first described as a proteolytic enzyme capable of degrading insulin (1). The enzyme is primarily located in the cytosol and peroxisomes (2), although its presence on the cell membrane (3, 4) and its secretion (5) have recently been reported. Although insulysin has the highest affinity for insulin, the enzyme has been shown to cleave a number of other physiological peptides in vitro including glucagon (6), insulin-like growth factors I and II (7), atrial natriuretic peptide (8), and transforming growth factor-␣ (9). The finding of a variety of substrates for the enzyme, as well as its presence at high levels in insulin-insensitive cells, suggest that insulysin has a variety of physiological functions. Those proposed include processing of insulin for antigen recognition (10), regulation of the multicatalytic proteinase (11), and modulation of steroid receptor action (12).Insulysin was shown to be identical to an enzymatic activity referred to as ␥-endorphin-generating enzyme, an enzyme that converts ␤-endorphin to ␥-endorphin (␤Ϫendorphin 1-17) and ␤Ϫendorphin 1-18 (13). In that study GRF, dynorphin B 1-13, dynorphin A 1-17, and pancreastatin 1-49 were shown also to be substrates. Recent interest in insulysin stems from its ability to degrade the amyloid peptides A␤ 1Ϫ40 and A␤ 1-42 (5, 14 -16) and its possible role as an enzyme involved in the clearance of amyloid peptides in the brain. Decreases in insulysin activity in the brains...

Substrate Activation of Insulin-degrading Enzyme (Insulysin)

“…Recombinant rat IDE was produced in Sf-9 cells as a fusion protein containing an N-terminal hexahistidine attached to the enzyme through a linker region containing a tobacco etch virus protease site (23). The enzyme was purified to homogeneity by chromatography on a His-Select HC nickel affinity gel column (Sigma).…”

Anion Activation Site of Insulin-degrading Enzyme

“…Enzyme Activity Assay-IDE activity was determined by measuring the increase in fluorescence that occurs when the enzyme cleaves the internally quenched fluorogenic substrate Abz-GGFLRKHGQ-EDDnp at the Arg-Lys bond (26). The reaction was followed on a SpectraMax Gemini XS fluorescence plate reader using excitation and emission wavelengths of 318 and 419 nm, respectively.…”

“…shown that the polyphosphate anions ATP and PPP i act in vitro as heterotropic activators of IDE toward the hydrolysis of small peptides, including the fluorogenic substrate Abz-GGFL-RKHGQ-EDDnp (21,26). To determine the interactions important for ATP binding in IDE, we crystallized rat IDE in the presence of ATP and determined the structure of the enzyme at 2.27 Å by molecular replacement using our previously determined native rat IDE coordinates (19).…”

“…Purification of IDE-IDE and the IDE K898A,K899A,S901A and IDE R429S mutants were expressed as hexahistidine fusion proteins (hexahistidine sequence and a linker containing a tobacco etch virus protease cleavage site) in Sf9 cells and purified as described previously (25,26). Briefly, Sf9 cells were suspended in 100 mM potassium phosphate buffer, pH 7.3, sonicated, and centrifuged at 75,000 ϫ g for 20 min to pellet the membrane fraction.…”

“…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. Bacmid and virus production were as discussed previously (25,26).…”