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

Arch Biochem Biophys. 2011 May 15;509(2):117-26. doi: 10.1016/j.abb.2011.03.006. Epub 2011 Mar 21.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetyl Coenzyme A / metabolism*
  • Adenosine Triphosphate / analogs & derivatives*
  • Adenosine Triphosphate / metabolism
  • Allosteric Regulation
  • Fluorescent Dyes / metabolism*
  • Kinetics
  • Magnesium Compounds / metabolism
  • Pyruvate Carboxylase / metabolism*
  • Rhizobium etli / enzymology*
  • Spectrometry, Fluorescence

Substances

  • Fluorescent Dyes
  • Magnesium Compounds
  • 2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate
  • Acetyl Coenzyme A
  • Adenosine Triphosphate
  • Pyruvate Carboxylase