A monomeric variant of insulin degrading enzyme (IDE) loses its regulatory properties

Abstract

Background: Insulin degrading enzyme (IDE) is a key enzyme in the metabolism of both insulin and amyloid beta peptides. IDE is unique in that it is subject to allosteric activation which is hypothesized to occur through an oligomeric structure.

Methodology/principal findings: IDE is known to exist as an equilibrium mixture of monomers, dimers, and higher oligomers, with the dimer being the predominant form. Based on the crystal structure of IDE we deleted the putative dimer interface in the C-terminal region, which resulted in a monomeric variant. Monomeric IDE retained enzymatic activity, however instead of the allosteric behavior seen with wild type enzyme it displayed Michaelis-Menten kinetic behavior. With the substrate Abz-GGFLRKHGQ-EDDnp, monomeric IDE retained approximately 25% of the wild type activity. In contrast with the larger peptide substrates beta-endorphin and amyloid beta peptide 1-40, monomeric IDE retained only 1 to 0.25% of wild type activity. Unlike wild type IDE neither bradykinin nor dynorphin B-9 activated the monomeric variant of the enzyme. Similarly, monomeric IDE was not activated by polyphosphates under conditions in which the activity of wild type enzyme was increased more than 50 fold.

Conclusions/significance: These findings serve to establish the dimer interface in IDE and demonstrate the requirement for an oligomeric form of the enzyme for its regulatory properties. The data support a mechanism where the binding of activators to oligomeric IDE induces a conformational change that cannot occur in the monomeric variant. Since a conformational change from a closed to a more open structure is likely the rate-determining step in the IDE reaction, the subunit induced conformational change likely shifts the structure of the oligomeric enzyme to a more open conformation.

Figure 1. Proposed dimer of IDE.

(A) Two orthogonal views of rIDE monomers from the crystal lattice are shown with elements making up the dimer interface highlighted in red for the top subunit. IDE is composed of four structurally related domains. Those domains are numbered in the top subunit in the left panel, and the N and C termini are indicated for the top subunit in the right panel. The C terminal region of IDE deleted in the rIDE^ΔC construct intended to destabilize the dimer interface is highlighted in blue for the lower monomers in both panels. (B) Close up of the dimer interface showing the region deleted in the rIDE^ΔC construct. Deleted residues 1002–1014 are shown in a stick representation with sheet-like hydrogen bonds across the interface at Leu¹⁰⁰⁴ indicated by dashed lines.

Figure 2. Comparison of the molecular weight forms of wild type IDE and IDE^ΔC on a Superdex S200 gel filtration column.

Wild type IDE or the IDE^ΔC variant was chromatographed on a Pharmacia Superdex S200 gel filtration column in 20 mM potassium phosphate buffer, pH 7.4. One ml of protein at 0.5 mg/ml was loaded and the column developed at a flow rate of 0.4 ml/min. Fractions were collected and assayed for IDE activity using Abz-GGFLRKHGQ-EDDnp as the substrate.

Figure 3. Comparison of the kinetics of IDE^ΔC and wild type IDE.

Reactions were conducted in 50 mM Tris-HCl, pH 7.4 using either 0.25 µg of wild type IDE (left) or 1.25 µg of IDE^ΔC (right) and Abz-GGFLRKHGQ-EDDnp as the variable substrate. Data were fit to either a hyperbolic or sigmoidal response curve as described in Methods with the values and errors included in Table 1.

Figure 4. HPLC chromatograms showing the cleavage of Aβ_1–40 and β-endorphin by IDE and IDE^ΔC.

(Top) Reaction mixtures containing 10 µM Aβ_1–40 in 50 mM Tris-HCl, pH 7.4 (curve A) were incubated with 100 ng of IDE for 5 min (curve B) or 2 µg of the monomeric IDE variant for 3 hrs (curve C). The amount of enzyme and time used was chosen so that a similar fraction of substrate would be consumed by both IDE and the monomeric IDE variant permitting a comparison of products formed at the same fraction of substrate consumed. Reaction products were separated by gradient HPLC on a Vydac C4 reverse phase column as previously described (16). Product peaks were collected manually and identified by mass spectrometry. Peak 1 is Aβ_1–14 (observed mass = 1698.72, calculated mass = 1698.72); peak 2 is Aβ_1–13 (observed mass = 1561.68, calculated mass = 1561.58); peak 3 is Aβ_1–18 (observed mass = 2167.32, calculated mass = 2167.32); peak 4 is Aβ_1–13 (observed mass = 2314.50, calculated mass = 2314.5); peak 5 is Aβ_1–20 (observed mass = 2461.67, calculated mass = 2461.67); peak 6 is Aβ_5–28 (observed mass = 1581.78, calculated mass = 1581.78); peak 7 is Aβ_21–40 (observed mass = 1885.96, calculated mass = 1886.2); peak 8 is Aβ_20–40 (observed mass = 2033.02, calculated mass = 2033.37); peak 9 is Aβ_4–40 (observed mass = 2785.50, calculated mass = 2786.29); peak 10 is Aβ_1–17 (observed mass = 2068.04, calculated mass = 2068.19); peak 11 is Aβ_26–40 (observed mass = 1413.80, calculated mass = 1414.73); peak 12 is Aβ_19–33 (observed mass = 1524.76, calculated mass = 1524.69); peak 13 is Aβ_14–22 (observed mass = 1118.60, calculated mass = 1118.3). Note peaks 3 and 4 are absent in the rIDE^ΔC cleavage products, while peaks 10 to 13 are unique to rIDE^ΔC. Although not shown the reaction was followed from ∼15% cleavage to ∼90% cleavage with similar results. (Bottom) Reaction mixtures containing 10 µM β-endorphin (βEp) in 50 mM Tris-HCl, pH 7.4 (curve A) were incubated with 50 ng of IDE for 15 min (curve B) or 2 µg of the monomeric IDE variant for 3 hrs (curve C). Reaction conditions and product separation and identification were as in (A) above. Peak 1 is β-endorphin 19–31 (observed mass = 1475.84, calculated mass = 1476.74); peak 2 is β-endorphin 18–31 (observed mass = 1623.91, calculated mass = 1623.91); peak 3 is β-endorphin 1–17 with methionine oxidized to its sulfoxide (observed mass = 1874.94, calculated mass = 1875.13); Peak 4 is β-endorphin 1–18 (observed mass = 2005.97, calculated mass = 2006.3); peak 5 is β-endorphin 7–20 (observed mass = 1590.86, calculated mass = 1591.82); peak 6 is β-endorphin 7–18 (observed mass = 1348.70, calculated mass = 1349.54). Note peaks 5 and 6 were only observed in the reaction with IDE^ΔC. Although not shown the reaction was followed from ∼20% cleavage to ∼85% cleavage with similar results.

Figure 5. Effect of bradykinin and dynorphin B9 on the reaction of IDE^ΔC and wild type IDE with Abz-GGFLRKHGQ-EDDnp.

Activity was determined in 50 mM Tris-HCl, pH 7.4 with 10 µM Abz-GGFLRKHGQ-EDDnp as substrate and the indicated concentrations of bradykinin (left) or dynorphin B9 (right). The reactions with wild type IDE (filled squares) contained 0.25 µg of protein and with IDE^ΔC (filled triangles) 1.25 µg of protein was used. With wild type IDE the maximal activation produced by bradykinin varied from 4.4 to 5.4 fold, while a 15 to 30% inhibition was seen with monomeric IDE. Similarly dynorphin B9 produced a 3.4 to 4.3 fold activation of wild type IDE, but inhibited the monomeric IDE variant 10 to 40%.

Figure 6. Effect of ATP and PPPi on the reaction of IDE^ΔC and wild type IDE with Abz-GGFLRKHGQ-EDDnp.

Reactions contained 50 mM Tris-HCl, pH 7.4 with 10 µM Abz-GGFLRKHGQ-EDDnp as substrate and the indicated concentrations of ATP (A) or PPPi (B). The reactions with wild type IDE (filled squares) contained 0.25 µg of protein while the reaction with IDE^ΔC (filled triangles) contained 1.25 µg of protein. In separate experiments ATP increased the activity of wild type IDE 130 to 160 fold, while PPPi increased the rate 88 to 97 fold. In contrast ATP increased the rate of the monomeric IDE variant 1.5 to 3 fold, while PPPi increased the rate 1.5 to 2.5 fold.

Figure 7. Binding of TNP-ATP to IDE^ΔC.

Fluorescence emission spectra of 10 µM TNP-ATP in the presence or absence of 1.5 µM of IDE^ΔC in 50 mM Tris-HCl, pH 7.4 were recorded on a Perkin-Elmer LS 55 Luminescence spectrometer. Fluorescence spectra were recorded with a λ_exc = 403 nm.