title: 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 beta 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 approximately 10-fold. Triphosphate and ATP shifted the oligomeric state of the enzyme from primarily dimer-tetramers to a monomer. These data suggest the presence of an allosteric regulatory site on insulysin that may shift its specificity toward small peptide substrates.
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0084, USA.
authors.forename: Eun Suk
authors.initials: ES
authors.lastname: Song
authors.forename: Maria Aparecida
authors.initials: MA
authors.lastname: Juliano
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Michael G
authors.initials: MG
authors.lastname: Fried
authors.forename: Steven L
authors.initials: SL
authors.lastname: Wagner
authors.forename: Louis B
authors.initials: LB
authors.lastname: Hersh
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000494:pharmacology*; D000495:Allosteric Site / Q000187:drug effects; D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D006868:Hydrolysis; D007339:Insulysin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D009711:Nucleotides / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D011122:Polyphosphates / Q000494:pharmacology*; D051381:Rats; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009711:Nucleotides; D009842:Oligopeptides; D010455:Peptides; D010710:Phosphates; D011122:Polyphosphates; D011994:Recombinant Proteins; C497348:abz-GGFLRKHGQ-EDDnp; D000255:Adenosine Triphosphate; D007339:Insulysin; C005692:triphosphoric acid