Location of lysine-beta 162 in mitochondrial F1-adenosinetriphosphatase.
The quenching of the fluorescence of bovine heart F1-adenosinetriphosphatase labeled specifically at its essential Lys-beta 162 with 7-chloro-4-nitro-2,1,3-benzoxadiazole (N-NBD-F1) by 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-triphosphate (TNP-ATP) has been studied. Analysis of the fluorescence data in the presence of 1 mM ATP shows that the dissociation constant of TNP-ATP from its first binding site in the covalently labeled enzyme is 250-fold lower than that of ATP, which it replaces in pH 7.0 buffer containing 25% glycerol, and that this binding causes a 54% quenching of the fluorescence of the N-NBD label due to energy transfer to the weakly fluorescent TNP-ATP molecule. Computation based on the observed quenching gives a distance of 25.6 +/- 0.4 A between the NBD label and the chromophore of the bound TNP-ATP molecule. Since the distance between the chromophore and the farthest O atom of the bound TNP-ATP is about 16 A, it seems quite likely that the epsilon-amino group of Lys-beta 162 is near the gamma-phosphate group of the TNP-ATP bound at the catalytic site. Similar measurements in the presence of 1 mM ADP show that the replacement of ADP at the catalytic site by TNP-ATP causes a 49% quenching of the fluorescence of the N-NBD label, which gives a distance of 26.5 +/- 0.4 A between the label and the chromophore of the bound TNP-ATP molecule.
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D007700:Kinetics; D008239:Lysine; D008433:Mathematics; D008929:Mitochondria, Heart / Q000201:enzymology*; D010069:Oxadiazoles / Q000494:pharmacology*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D010069:Oxadiazoles; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan; D008239:Lysine