Detecting precatalytic conformational changes in F1-ATPase with 4-benzoyl(benzoyl)-1-amidofluorescein, a novel fluorescent nucleotide site-specific photoaffinity label.
A novel photoaffinity label for studies with the F1-ATPase has been synthesized and found to be an effective reporter of subunit conformational changes that occur in this enzyme upon multiple nucleotide-binding site occupancy. The new probe, 4-benzoyl(benzoyl)-1-amidofluorescein (BzAF), which possesses structural similarity to purine nucleotides, exhibits bifunctional characteristics that enable it to bind covalently to the exchangeable nucleotide sites on beef heart F1 (via photoactivation of the benzophenone moiety) and, once covalently linked, emit environmentally sensitive fluorescence (via selective excitation of the fluorescein moiety). BzAF binds competitively with ATP in the absence of illumination, with a KI of 50 microM. Under actinic irradiation necessary for generating the covalently reacting diradical triplet state of benzophenone, BzAF behaves as a nucleotide site-directed photoaffinity label of exchangeable (catalytic) sites, and the resulting photoinhibition of ATPase activity displays pseudo first-order rate-saturation kinetics that support formation of a dissociable BzAF.F1 complex (k-1/k1 = 58 microM) prior to covalent binding. The BzAF-induced photoinactivation is protectable with native nucleotide ligand (e.g. MgADP, Kprotect = 0.4 mM). Added corroboration of a catalytic cooperativity mechanism for F1 was obtained by finding a molar stoichiometric ratio [( 3H]BzAF:F1) of 1 required for complete inhibition of ATPase activity. Steady-state fluorescence studies with a unisite-labeled BzAF.F1 complex (a catalytically inactive species on which at least one exchangeable nucleotide-binding site remains unoccupied) display a saturable fluorescence quenching of the bound fluorescein upon titration with MgADP, but no change with MgAMP. These data imply that the filling of more than one of the catalytic binding sites/mol of F1 with nucleotide signals a precatalytic conformational adjustment that is transmitted between catalytic sites and across the beta-alpha-beta domain of the enzyme's subunit structure.
Pal
P K
PK
Department of Biology, New York University, New York 10003.
Coleman
P S
PS
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D005452:Fluoresceins / Q000138:chemical synthesis / Q000378:metabolism*; D005456:Fluorescent Dyes; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D015846:Monokines / Q000235:genetics*; D009206:Myocardium / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D000345:Affinity Labels; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015846:Monokines; C065577:4-benzoyl(benzoyl)-1-amidofluorescein; D006180:Proton-Translocating ATPases