Characterization of the binding of the fluorescent ATP analog TNP-ATP to insulysin

Hongbing Yao, Louis B. Hersh

Research output: Contribution to journalArticlepeer-review

21 Scopus citations


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 (KD) of TNP-ATP was determined as 1.15 μM, while the activation constant (KA) was determined to be 1.6 μM. Competition experiments were used to show that ATP (Ki = 1.3 mM) and PPPi (Ki = 0.9 mM) bind with a higher affinity than ADP (2.2 mM) and AMP (4.0 mM). Adenosine did not bind to the anion binding site.

Original languageEnglish
Pages (from-to)175-181
Number of pages7
JournalArchives of Biochemistry and Biophysics
Issue number2
StatePublished - Jul 15 2006

Bibliographical note

Funding Information:
This work was supported in part by Grants DA 02243 from the National Institute on Drug Abuse, AG 24899 from the National Institute on Aging, NS 46517 from the National Institute on Neurological Disorders and Stroke, and P20 RR02017 from the NCRR.


  • ATP-analog
  • Allosteric site
  • Binding
  • Peptidase
  • kinetics

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology


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