ATP Effects on Insulin-degrading Enzyme Are Mediated Primarily through Its Triphosphate Moiety

Abstract

It has been reported previously that ATP inhibits the insulysin reaction (Camberos, M. C., Perez, A. A., Udrisar, D. P., Wanderley, M. I., and Cresto, J. C. (2001) Exp. Biol. Med. 226, 334 -341). We report here that with 2-aminobenzoyl-GGFLRKHGQ-ethylenediamine-2,4-dinitrophenyl as substrate, ATP and other nucleotides increase the rate >20-fold in Tris buffer. There is no specificity with respect to the nucleotide; however, ATP is more effective than ADP, which is more effective than AMP. Triphosphate itself was as effective as ATP, indicating it is this moiety that is responsible for activation. The binding of triphosphate was shown to be at a site distinct from the active site, thus acting as a noncompetitive activator. With the physiological substrates insulin and amyloid ␤ peptide, nucleotides and triphosphate were without effect. However, with small physiological peptides such as bradykinin and dynorphin B-9, ATP and triphosphate increased the rate of hydrolysis ϳ10-fold. Triphosphate and ATP shifted the oligomeric state of the enzyme from primarily dimertetramers to a monomer. These data suggest the presence of an allosteric regulatory site on insulysin that may shift its specificity toward small peptide substrates.Insulysin (insulin-degrading enzyme (IDE), 1 EC 3.4.24.56) in conjunction with neprilysin, endothelin-converting enzyme, plasmin, and possibly other peptidases provides a major catabolic pathway for amyloid ␤ peptides in the brain (1-3). A decrease in the activity of one or more of these peptidases would lead to increased A␤ and its subsequent oligomerization and deposition. The most convincing evidence that IDE participates in A␤ clearance is the finding of elevated levels of both A␤ 1-40 and A␤ 1-42 in the brains of mice in which the IDE gene was disrupted (4, 5). Recent genetic evidence suggests, but has yet to be firmly established, that IDE is linked to late onset Alzheimer's disease (6 -8). Any such linkage between IDE and late onset Alzheimer's disease is likely through the ability of IDE to degrade amyloid ␤ peptides.We recently reported that IDE displays the characteristics of an allosteric enzyme, whereby peptide substrates increase the rate of hydrolysis of other peptide substrates 2-7-fold (9). This behavior could be attributed to two effects as follows: a peptidedependent shift in the monomer-oligomer equilibrium of IDE to favor a dimer, and a conformational change produced by the binding of peptide to one subunit that increased the activity of the adjacent subunit.The finding that IDE displays allosteric properties led us to investigate further the reported effect of ATP and other nucleotides acting as noncompetitive inhibitors of the IDE-dependent hydrolysis of insulin (10). The results of this study show that nucleotide triphosphates can increase the rate of cleavage of small peptide substrates of IDE, and that the effect of nucleotide triphosphates can be attributed primarily to the triphosphate moiety. Triphosphates bind to a site distinct from the substrate-binding site. Thus ...

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

“…The IDE activity in cells may also be regulated by non-esterified fatty acids [34], nucleotide triphosphates [35] and post-translational modification [36]. In particular, ATP induces conformational and aggregation changes in IDE and inhibits insulin degradation in vitro [37].…”

Nitric oxide inhibits insulin-degrading enzyme activity and function through S-nitrosylation

“…Camberos et al showed ATP inhibited insulin degradation [15]. However, it has also been reported that ATP can increase degradation of small peptides by IDE, while having little effect on insulin metabolism [16,17]. Whatever the effect on insulin degradation, ATP does induce a conformational change in IDE [16].…”

Anion Activation Site of Insulin-degrading Enzyme

“…The enzyme was maintained for 4 days under the above conditions before being used for crystallization trials employing the sitting drop vaporization method with Compact Clover plates (Emerald Biosystems). EDTA was used primarily to sequester any free Mg 2ϩ , which has been shown to reduce activation by ATP (21,23). The protein/ATP solution was mixed 1:1 with well solution containing 100 mM sodium citrate, pH 6.5, 100 mM ammonium acetate, and 20% PEG 4000, and crystals were grown at 22°C, usually accompanied by heavy precipitation.…”

“…In addition, IDE has been shown to be activated heterotropically by polyphosphate anions (21,22). In particular, ATP, other nucleotides, and triphosphate (PPP i ) activate the enzyme toward hydrolysis of certain substrates 100 -200-fold (21,23). More recently, it has been reported that IDE has ATPase activity, which is also dependent upon a site distinct from the active site (24).…”

“…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). In particular, ATP, other nucleotides, and triphosphate (PPP i ) activate the enzyme toward hydrolysis of certain substrates 100 -200-fold (21,23).…”