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Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA
Authors: Abdussalam Adina-Zada
Rasmani Hazra
Chutima Sereeruk
Sarawut Jitrapakdee
Tonya N. Zeczycki
Martin St Maurice
W. Wallace Cleland
John C. Wallace
Paul V. Attwood
University of Western Australia
Mahidol University
University of Wisconsin Madison
Marquette University
University of Adelaide
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 15-May-2011
Citation: Archives of Biochemistry and Biophysics. Vol.509, No.2 (2011), 117-126
Abstract: 2′,3′-O-(2,4,6-Trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) is a fluorescent analogue of ATP. MgTNP-ATP was found to be an allosteric activator of pyruvate carboxylase that exhibits competition with acetyl CoA in activating the enzyme. There is no evidence that MgTNP-ATP binds to the MgATP substrate binding site of the enzyme. At concentrations above saturating, MgATP activates bicarbonate-dependent ATP cleavage, but inhibits the overall reaction. The fluorescence of MgTNP-ATP increases by about 2.5-fold upon binding to the enzyme and decreases on addition of saturating acetyl CoA. However, not all the MgTNP-ATP is displaced by acetyl CoA, or with a combination of saturating concentrations of MgATP and acetyl CoA. The kinetics of the binding of MgTNP-ATP to pyruvate carboxylase have been measured and shown to be triphasic, with the two fastest phases having pseudo first-order rate constants that are dependent on the concentration of MgTNP-ATP. The kinetics of displacement from the enzyme by acetyl CoA have been measured and also shown to be triphasic. A model of the binding process is proposed that links the kinetics of MgTNP-ATP binding to the allosteric activation of the enzyme. © 2011 Elsevier Inc. All rights reserved.
ISSN: 10960384
Appears in Collections:Scopus 2011-2015

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