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 concentratio ns did not prevent binding and hydrolysis of submicromolar concentrati on of trinitrophenyl adenosine triphosphate (Tnp-ATP) by F-1-ATPase [M urataliev, M. B. & Boyer, P. O. (1994) J. Biol. Chem. 269, 15431-15439 ]. To explore F-1-ATPase binding sites that bind Tnp-ATP a new photore active analog of ATP, 2-azido-trinitrophenyl adenosine triphosphate (2 -N-3-Tnp-ATP) has been synthesized and used for photoaffinity labeling of mitochondrial F-1-ATPase. The analog shares many properties of the parent non-azido Tnp-ATP as shown from spectral characteristics, bind ing with F-1-ATPase, and kinetic and inhibition studies, 500 mu M ATP does not prevent binding and hydrolysis of low concentrations of 2-N-3 -Tnp-ATP by F-1-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 in itial binding at a third catalytic site. The results give strong evide nce that only two catalytic sites need to have bound substrate for nea r maximal turnover rate, and that three catalytic sites of F-1-ATPase participate equally in catalysis. When F-1-ATPase binds substoichiomet ric 2-N-3-Tnp-ATP in the presence of Mg2+, illumination of the inactiv e complex formed results in the covalent labeling of a catalytic site. This shows that F-1-ATPase forms similar inactive complexes when ADP or Tnp-ADP is bound at a catalytic site in the presence of Mg2+. Expos ure of the nucleotide-depleted F-1-ATPase to 20 mu M 2-N-3-Tnp-ATP fol lowed by a short incubation with excess of Tnp-ATP results in binding, and, upon illumination, in a covalent labeling of a non-catalytic-sir e peptide.