Chemical evidence for probably nonequivalent beta subunits in F1 adenosinetriphosphatase.
Mitochondrial F1 adenosinetriphosphatase (MF1) was allowed to react with 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl) until its NBD label to MF1 molar ratio (n) reached approximately 2.6. The labeled enzyme was then separated and subsequently allowed to react with dithiothreitol (DTT) in a controlled way to have its covalent label partially removed. At various stages of removal of its covalent label, the enzyme was separated and assayed for its value of n and the ratio (r) of specific ATPase activity of the DDT-treated enzyme to that of the unlabeled control sample. Most of the experimental values of r turned out to be significantly higher than the theoretical maximum values for models of the enzyme with three equivalent beta subunits, which have been shown to be equal to (1 - n/3)3 for three alternating sites and (1 - n/3)2 for two alternating sites. On the other hand, the observed values of n and r are consistent with a model of the enzyme based on nonequivalent beta subunits, with one active catalytic site and two latent catalytic sites that normally have only regulatory function. Possible complication in the interpretation of data due to a significant amount of nonspecific labeling by NBD-Cl has also been examined and discussed. In addition, the NBD label has been used, after its transfer from the essential Tyr to the essential Lys, as an internal fluorescent probe to monitor protein conformation change at the active site of MF1. Experimental data show that the binding of adenine nucleotide at the latent site(s) can cause conformational change at the active site and presumably in this way regulate the catalytic property of the active site.
D009327:4-Chloro-7-nitrobenzofurazan; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004229:Dithiothreitol; D008929:Mitochondria, Heart / Q000201:enzymology; D011487:Protein Conformation; D006180:Proton-Translocating ATPases
D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan; D004229:Dithiothreitol