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

Abdussalam Adina-ZadaRasmani HazraChutima SereerukSarawut JitrapakdeeTonya N. ZeczyckiMartin St MauriceW. Wallace ClelandJohn C. WallacePaul V. AttwoodUniversity of Western AustraliaMahidol UniversityUniversity of Wisconsin MadisonMarquette UniversityUniversity of Adelaide2018-05-032018-05-032011-05-15Archives of Biochemistry and Biophysics. Vol.509, No.2 (2011), 117-12610960384000398612-s2.0-79955057247https://repository.li.mahidol.ac.th/handle/123456789/115522′,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.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyProbing 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 CoAArticleSCOPUS10.1016/j.abb.2011.03.006