title: Characterization of the binding of the fluorescent ATP analog TNP-ATP to insulysin.
abstract: It has recently been reported that insulin-degrading enzyme (IDE) contains an allosteric site which binds polyanions such as ATP and PPPi. This site is distinct from the catalytic site where homotrophic allosteric effects are produced. In this study, we have characterized the binding of ATP to this anion binding site using the fluorescent ATP analog 2',3'-O-(2,4,6-trinitrophenyl)-adenosine triphosphate (TNP-ATP), which exhibits a higher affinity to the enzyme than ATP itself. TNP-ATP binding to IDE was accompanied by a more than 4-fold increase in fluorescence. The dissociation constant (K(D)) of TNP-ATP was determined as 1.15 microM, while the activation constant (K(A)) was determined to be 1.6 microM. Competition experiments were used to show that ATP (Ki = 1.3 mM) and PPPi (Ki = 0.9mM) bind with a higher affinity than ADP (2.2 mM) and AMP (4.0 mM). Adenosine did not bind to the anion binding site.
authors.affiliation: Department of Molecular and Cellular Biochemistry and the Center for Structural Biology, University of Kentucky, Lexington, KY 40536-0509, USA.
authors.forename: Hongbing
authors.initials: H
authors.lastname: Yao
authors.forename: Louis B
authors.initials: LB
authors.lastname: Hersh
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D001667:Binding, Competitive; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007339:Insulysin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010710:Phosphates / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D018411:Spodoptera / Q000166:cytology; D013379:Substrate Specificity; D013388:Succinimides / Q000494:pharmacology
chemical_list: D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D007004:Hypoglycemic Agents; D007328:Insulin; D010710:Phosphates; D011993:Recombinant Fusion Proteins; D013388:Succinimides; D006639:Histidine; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D007339:Insulysin; C019358:disuccinimidyl suberate