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.
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.
authors.affiliation: School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley, WA 6009, Australia.
authors.forename: Abdussalam
authors.initials: A
authors.lastname: Adina-Zada
authors.forename: Rasmani
authors.initials: R
authors.lastname: Hazra
authors.forename: Chutima
authors.initials: C
authors.lastname: Sereeruk
authors.forename: Sarawut
authors.initials: S
authors.lastname: Jitrapakdee
authors.forename: Tonya N
authors.initials: TN
authors.lastname: Zeczycki
authors.forename: Martin
authors.initials: M
authors.lastname: St Maurice
authors.forename: W Wallace
authors.initials: WW
authors.lastname: Cleland
authors.forename: John C
authors.initials: JC
authors.lastname: Wallace
authors.forename: Paul V
authors.initials: PV
authors.lastname: Attwood
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000494:Allosteric Regulation; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D017616:Magnesium Compounds / Q000378:metabolism; D011766:Pyruvate Carboxylase / Q000378:metabolism*; D042163:Rhizobium etli / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D017616:Magnesium Compounds; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000105:Acetyl Coenzyme A; D000255:Adenosine Triphosphate; D011766:Pyruvate Carboxylase