The plasma membrane H+ -ATPase, a simple polypeptide with a long history.

The plasma membrane H+ -ATPase of fungi and plants is a single polypeptide of fewer than 1,000 residues that extrudes protons from the cell against a large electric and concentration gradient. The minimalist structure of this nanomachine is in stark contrast to that of the large multi-subunit FO F1 ATPase of mitochondria, which is also a proton pump, but under physiological conditions runs in the reverse direction to act as an ATP synthase. The plasma membrane H+ -ATPase is a P-type ATPase, defined by having an obligatory phosphorylated reaction cycle intermediate, like cation pumps of animal membranes, and thus, this pump has a completely different mechanism to that of FO F1 ATPases, which operates by rotary catalysis. The work that led to these insights in plasma membrane H+ -ATPases of fungi and plants has a long history, which is briefly summarized in this review.

Two ATPases.

In this article, I reflect on research on two ATPases. The first is F(1)F(0)-ATPase, also known as ATP synthase. It is the terminal enzyme in oxidative phosphorylation and famous as a nanomotor. Early work on mitochondrial enzyme involved purification in large amount, followed by deduction of subunit composition and stoichiometry and determination of molecular sizes of holoenzyme and individual subunits. Later work on Escherichia coli enzyme utilized mutagenesis and optical probes to reveal the molecular mechanism of ATP hydrolysis and detailed facets of catalysis. The second ATPase is P-glycoprotein, which confers multidrug resistance, notably to anticancer drugs, in mammalian cells. Purification of the protein in large quantity allowed detailed characterization of catalysis, formulation of an alternating sites mechanism, and recently, advances in structural characterization.

ATP synthases in the year 2000: defining the different levels of mechanism and getting a grip on each.

ATP synthases are unusually complex molecules, which fractionate most readily into two major units, one a water soluble unit called F(1) and the other a detergent soluble unit called F(0). In almost all known species the F(1) unit consists of 5 subunit types in the stoichiometric ratio alpha(3)beta(3)gammadeltaepsilon while the F(0) unit contains 3 subunit types (a, b, and c) in E. coli, and at least 10 subunit types (a, b, c, and others) in higher animals. It is now believed by many investigators that during the synthesis of ATP, protons derived from an electrochemical gradient generated by an electron transport chain are directed through the F(0) unit in such a way as to drive the rotation of the single gamma subunit, which extends from an oligomeric ring of at least 10 c subunits in F(0) through the center of F(1). It is further believed by many that the rotating gamma subunit, by interacting sequentially with the 3 alphabeta pairs of F(1) (360 degrees cycle) in the presence of ADP, P(i), and Mg++, brings about via "power strokes" conformational/binding changes in these subunits that promote the synthesis of ATP and its release on each alphabeta pair. In support of these views, studies in several laboratories either suggest or demonstrate that F(0) consists in part of a proton gradient driven motor while F(1) consists of an ATP hydrolysis driven motor, and that the gamma subunit does rotate during F(1) function. Therefore, current implications are that during ATP synthesis the former motor drives the latter in reverse via the gamma subunit. This would suggest that the process of understanding the mechanism of ATP synthases can be subdivided into three major levels, which include elucidating those chemical and/or biophysical events involved in (1) inducing rotation of the gamma subunit, (2) coupling rotation of this subunit to conformational/binding changes in each of the 3 alphabeta pairs, and (3) forming ATP and water (from ADP, P(i), and Mg(++)) and then releasing these products from each of the 3 catalytic sites. Significantly, it is at the final level of mechanism where the bond breaking/making events of ATP synthesis occur in the transition state, with the former two levels of mechanism setting the stage for this critical payoff event. Nevertheless, in order to get a better grip in this new century on how ATP synthases make ATP and then release it, we must take on the difficult challenge of elucidating each of the three levels of mechanism.

Stoichiometry of energy coupling by proton-translocating ATPases: a history of variability.

One of the central energy-coupling reactions in living systems is the intraconversion of ATP with a transmembrane proton gradient, carried out by proton-translocating F- and V-type ATPases/synthases. These reversible enzymes can hydrolyze ATP and pump protons, or can use the energy of a transmembrane proton gradient to synthesize ATP from ADP and inorganic phosphate. The stoichiometry of these processes (H(+)/ATP, or coupling ratio) has been studied in many systems for many years, with no universally agreed upon solution. Recent discoveries concerning the structure of the ATPases, their assembly and the stoichiometry of their numerous subunits, particularly the proton-carrying proteolipid (subunit c) of the F(O) and V(0) sectors, have shed new light on this question and raise the possibility of variable coupling ratios modulated by variable proteolipid stoichiometries.