Evidence that the Mg-dependent low-affinity binding site for ATP and Pi demonstrated on the isolated beta subunit of the F0.F1 ATP synthase is a catalytic site.

Abstract

complex is still unknown, although a number of mechanisms have been proposed (6, 7). Elucidation of the mechanism of action of this enzyme complex requires a detailed description of its catalytic site. This includes identification of the catalytic subunit and characterization of substrate binding sites and essential amino acid residues on this subunit.A large number of studies using different approaches point to the p subunit as the one that contains the catalytic site and is involved in substrate binding (3,4,8). Thus, various studies have shown that the F1 sector has several nucleotide binding sites that reside in its two larger subunits, a and /3(4, 7, 9-11). At least one Pi binding site has also been revealed in the F1 sector (12, 13), and from studies with a Pi analog it seems to be located on the p subunit (14-16). Attempts to identify functional amino acids in the catalytic site have been carried out by using labeled chemical modifiers, known to interact with specific amino acid residues, that bind to the F1 ATPase and inactivate it. After dissociating the labeled enzyme complex to its individual subunits, the label was detected mainly on the p subunit (7,8).A detailed characterization of individual substrate binding sites and their possible identification with the catalytic site in the Fl-enzyme complex is difficult because of the complexity of its structure and function. It contains two or three copies of aB pairs, which could be in different conformational states in the catalytically active complex (17,18), and its activity leads to interconversion of the substrates. These difficulties may be overcome if, instead of testing the whole F1 sector, isolated, purified, and reconstitutively active a and p subunits are used. Such subunits have been obtained so far only from three bacterial sources: a thermophilic bacterium (19), Escherichia coli (20), and Rhodospirillum rubrum (21). They are indeed a much simpler system for study as they show no subunit-subunit interactions and no catalytic activity by themselves, although they can restore ATP synthesis or hydrolysis (or both) to preparations that lack them (19-21).Direct binding studies with labeled substrates have been carried out only with the a subunit of E. coli (22) and the 8 subunit of R. rubrum (23,24,41). These studies have identified one nucleotide binding site on the a subunit (22), whereas the p subunit contained two binding sites for ATP (23) or ADP (24) and one binding site for Pi (41). One of the nucleotide binding sites on the p subunit is a Mg-dependent high-affinity site, which is very similar to the binding site located on the a subunit. The second is a Mg-dependent low-affinity site, which has been suggested to contain also the binding site for Pi, because both nucleotides have been found to be competitive inhibitors of Pi binding (41). Furthermore, Pi seems to bind at the site occupied by the yphosphate group of ATP, since ATP is a much more potent inhibitor of Pi binding to the p subunit than is ADP.In this study we attempt to identify ...

ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas

“…Diethyl pyrocarbonate modifies the ATP synthase ␤ subunit, completely preventing the binding of phosphate. It also blocks the binding of ATP to a Mg 2ϩ -dependent low-affinity site (203,381,445). In contrast, the ADP binding capacity of the ␤ subunit is not (203).…”

“…It also blocks the binding of ATP to a Mg 2ϩ -dependent low-affinity site (203,381,445). In contrast, the ADP binding capacity of the ␤ subunit is not (203). Diethyl pyrocarbonate also modifies F 0 from E. coli, inducing inhibition of proton uptake (381).…”

“…The chemical modification of the ␤ subunit of F 1 from Rhodospirillum rubrum with this reagent results in loss of both phosphate and ATP binding capacities (203). However, ADP binding sites remain active.…”

Binding energy, conformational change, and the mechanism of transmembrane solute movements.

“…These enzymes differ from the proton pumps of the aspartyl-phosphoryl-enzyme intermediate family of transport ATPases in that they do not have a covalent phosphoryl-enzyme intermediate (186), and they are also structurally more complex, with multiple cooperatively interacting hydrolytic subunits and a separate protonconducting channel. These features no doubt contribute to the ability of these enzymes to efficiently perform their dual physiological roles of ATP-dependent proton translocation and ATP synthesis in response6to a transmembrane electrochemical proton gradient in a rapid and regulated manner; nevertheless, the molecular events occurring at the individual active sites of these enzymes (possibly in an interdomain cleft in each ,B subunit [79]) may be fundamentally quite similar to those of the aspartyl-phosphoryl-enzyme intermediate family, as the diagram intends to show. Since there is no covalent intermediate, the condensation reaction for these pumps involves only ATP (or Mg ATP) binding as depicted in stages 1 and 2.…”

Differentiation of mucous neck cells into parietal cells: a new concept of mitochondrial biogenesis

“…V (vacuolar) ATPases are related to the acidification of clathrin vesicles (Xie and Stone, 1986;Brown et al, 1987), Golgi complex (Araki et al, 1990), and lysosomes (Moriyama and Nelson, 1989). F (coupling factor) ATPases are localised on the mitochondrial internal membrane (Khananshvili and Gromet-Helhanan, 1985). P (plasma) ATPases, detected by potassiumdependent p-nitrophenylphosphatase (K + -pNPPase) activity (Mayahara et al, 1980), are present in plasma membrane of cells of different organs, for example, in kidney (Mayahara et al, 1982) or central nervous system (Inomata et al, 1983).…”