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

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.

► MgTNP-ATP is not a competitive inhibitor of pyruvate carboxylase versus MgATP.
► MgTNP-ATP activates the enzyme similarly to the allosteric activator, acetyl CoA.
► MgTNP-ATP binds to nucleotide binding sites on catalytically inactive subunits.
► The kinetics of MgTNP-ATP binding are triphasic.
► High [MgATP] allosterically activates the bicarbonate-dependent ATP cleavage.