The presence of two hydrolytic sites on beef heart mitochondrial adenosine triphosphatase.
The ribose-modified nucleotides 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) and TNP-ADP were used to probe the catalytic sites on soluble beef heart mitochondrial adenosine triphosphatase (F1). Both compounds were potent competitive inhibitors of ATP hydrolysis catalyzed by F1, Ki = 5.5 and 10 nM, respectively, and by submitochondrial particles, Ki (TNP-ATP) = 21 nM. Both compounds also were potent competitive inhibitors of ATP synthesis during oxidative phosphorylation (Ki = 1300 nM). Both analogs inhibited the 32Pi-ATP exchange reaction and the ATP-dependent reduction of NAD+ by succinate, catalyzed by submitochondrial particles. TNP-ATP and TNP-ADP were bound by F1. The presence of two binding sites on the enzyme for TNP-adenine nucleotides was determined by titrations of difference absorbance spectra, of the increase in fluorescence of the analog which occurred upon interaction with protein, and by titrations with the centrifuge column method using 32P-labeled TNP-adenine nucleotides. The first binding site bound the analogs with an affinity too high to be measured. The Kd for analog binding by the second site was 20 to 80 nM. In the presence of Mg2+, the 2 sites were filled with the TNP-ATP at a rate too rapid to be resolved by the procedure used. TNP-[gamma-32P]ATP was hydrolyzed by F1, Km = 0.2 microM, Vmax = 1.1 mol of 32Pi formed/mol of F1/s. It was shown, using the isotope trap technique as well as the inhibitor efrapeptin, that the 2 binding sites for TNP-ATP on F1 are hydrolytic sites.
D000227:Adenine Nucleotides; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D006868:Hydrolysis; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D010086:Oxidative Phosphorylation Coupling Factors / Q000378:metabolism*; D011485:Protein Binding; D006180:Proton-Translocating ATPases; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014301:Trinitrobenzenes
D000227:Adenine Nucleotides; D010086:Oxidative Phosphorylation Coupling Factors; D014301:Trinitrobenzenes; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases