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.
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.
Murataliev
M B
MB
Molecular Biology Institute, University of California, Los Angeles, USA.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D010782:Photolysis; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013053:Spectrophotometry
C095967:2-azido-2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D000345:Affinity Labels; D004791:Enzyme Inhibitors; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases