Chemical evidence for probably nonequivalent .beta. subunits in F1 adenosine triphosphatase

Publisher Website
Google Scholar
Abstract
Bovine heart mitochondrial F1 ATPase (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 .apprx. 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 DTT-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 3 equivalent .beta. subunits, which were shown to be equal to (1-n/3)3 for 3 alternating sites and (1-n/3)2 for 2 alternating sites. The observed values of n and r are consistent with a model of the enzyme based on nonequivalent .beta. subunits, with 1 active catalytic site and 2 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 was also examined and discussed. In addition, the NBD label was 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.