Interaction of mitochondrial F1-ATPase with trinitrophenyl derivatives of ATP. Photoaffinity labeling of binding sites with 2-azido-2',3'-O-(4,6-trinitrophenyl)adenosine 5'-triphosphate

Eur J Biochem. 1995 Sep 1;232(2):578-85. doi: 10.1111/j.1432-1033.1995.tb20847.x.


It was shown recently that ATP present at near saturating concentrations did not prevent binding and hydrolysis of submicromolar concentration of trinitrophenyl adenosine triphosphate (Tnp-ATP) by F1-ATPase [Murataliev, M. B. & Boyer, P. O. (1994) J. Biol. Chem. 269, 15431-15439]. To explore F1-ATPase binding sites that bind Tnp-ATP a new photoreactive analog of ATP, 2-azido-trinitrophenyl adenosine triphosphate (2-N3-Tnp-ATP) has been synthesized and used for photoaffinity labeling of mitochondrial F1-ATPase. The analog shares many properties of the parent non-azido Tnp-ATP as shown from spectral characteristics, binding with F1-ATPase, and kinetic and inhibition studies. 500 microM ATP does not prevent binding and hydrolysis of low concentrations of 2-N3-Tnp-ATP by F1-ATPase. Photoirradiation of the enzyme-analog complex formed under such conditions results in the labeling of the catalytic-site peptide. This shows that in the presence of near saturating ATP, Tnp-ATP can enter the catalytic cycle and inhibit ATP hydrolysis by initial binding at a third catalytic site. The results give strong evidence that only two catalytic sites need to have bound substrate for near maximal turnover rate, and that three catalytic sites of F1-ATPase participate equally in catalysis. When F1-ATPase binds substoichiometric 2-N3-Tnp-ATP in the presence of Mg2+, illumination of the inactive complex formed results in the covalent labeling of a catalytic site. This shows that F1-ATPase forms similar inactive complexes when ADP or Tnp-ADP is bound at a catalytic site in the presence of Mg2+. Exposure of the nucleotide-depleted F1-ATPase to 20 microM 2-N3-Tnp-ATP followed by a short incubation with excess of Tnp-ATP results in binding, and, upon illumination, in a covalent labeling of a non-catalytic-site peptide.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives*
  • Adenosine Triphosphate / chemistry
  • Adenosine Triphosphate / metabolism
  • Adenosine Triphosphate / pharmacology
  • Affinity Labels / chemistry
  • Affinity Labels / metabolism*
  • Affinity Labels / pharmacology
  • Animals
  • Binding Sites
  • Cattle
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / metabolism
  • Enzyme Inhibitors / pharmacology
  • In Vitro Techniques
  • Kinetics
  • Mitochondria, Heart / enzymology*
  • Photolysis
  • Proton-Translocating ATPases / antagonists & inhibitors
  • Proton-Translocating ATPases / metabolism*
  • Spectrophotometry


  • 2-azido-2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate
  • Affinity Labels
  • Enzyme Inhibitors
  • Adenosine Triphosphate
  • Proton-Translocating ATPases