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

Hongbing Yao; Louis B. Hersh

doi:10.1016/j.abb.2006.04.011

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

Hongbing Yao, Louis B. Hersh

Research output: Contribution to journal › Article › peer-review

21 Scopus citations

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 μM, while the activation constant (K_A) was determined to be 1.6 μM. Competition experiments were used to show that ATP (K_i = 1.3 mM) and PPPi (K_i = 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 language	English
Pages (from-to)	175-181
Number of pages	7
Journal	Archives of Biochemistry and Biophysics
Volume	451
Issue number	2
DOIs	https://doi.org/10.1016/j.abb.2006.04.011
State	Published - 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.

Keywords

ATP-analog
Allosteric site
Binding
Peptidase
kinetics

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology

Access to Document

10.1016/j.abb.2006.04.011

Cite this

@article{1d25f883e8db4fc0a5f193650044eb79,

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 (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.",

keywords = "ATP-analog, Allosteric site, Binding, Peptidase, kinetics",

author = "Hongbing Yao and Hersh, {Louis B.}",

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.",

year = "2006",

month = jul,

day = "15",

doi = "10.1016/j.abb.2006.04.011",

language = "English",

volume = "451",

pages = "175--181",

number = "2",

}

TY - JOUR

T1 - Characterization of the binding of the fluorescent ATP analog TNP-ATP to insulysin

AU - Yao, Hongbing

AU - Hersh, Louis B.

N1 - 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.

PY - 2006/7/15

Y1 - 2006/7/15

N2 - 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.

AB - 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.

KW - ATP-analog

KW - Allosteric site

KW - Binding

KW - Peptidase

KW - kinetics

UR - http://www.scopus.com/inward/record.url?scp=33745713815&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33745713815&partnerID=8YFLogxK

U2 - 10.1016/j.abb.2006.04.011

DO - 10.1016/j.abb.2006.04.011

M3 - Article

C2 - 16723115

AN - SCOPUS:33745713815

VL - 451

SP - 175

EP - 181

IS - 2

ER -

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

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this