Rotary Ion-Translocating ATPases/ATP Synthases: Diversity, Similarities, and Differences.

Ion-translocating ATPases and ATP synthases (F-, V-, A-type ATPases, and several P-type ATPases and ABC-transporters) catalyze ATP hydrolysis or ATP synthesis coupled with the ion transport across the membrane. F-, V-, and A-ATPases are protein nanomachines that combine transmembrane transport of protons or sodium ions with ATP synthesis/hydrolysis by means of a rotary mechanism. These enzymes are composed of two multisubunit subcomplexes that rotate relative to each other during catalysis. Rotary ATPases phosphorylate/dephosphorylate nucleotides directly, without the generation of phosphorylated protein intermediates. F-type ATPases are found in chloroplasts, mitochondria, most eubacteria, and in few archaea. V-type ATPases are eukaryotic enzymes present in a variety of cellular membranes, including the plasma membrane, vacuoles, late endosomes, and trans-Golgi cisternae. A-type ATPases are found in archaea and some eubacteria. F- and A-ATPases have two main functions: ATP synthesis powered by the proton motive force (pmf) or, in some prokaryotes, sodium-motive force (smf) and generation of the pmf or smf at the expense of ATP hydrolysis. In prokaryotes, both functions may be vitally important, depending on the environment and the presence of other enzymes capable of pmf or smf generation. In eukaryotes, the primary and the most crucial function of F-ATPases is ATP synthesis. Eukaryotic V-ATPases function exclusively as ATP-dependent proton pumps that generate pmf necessary for the transmembrane transport of ions and metabolites and are vitally important for pH regulation. This review describes the diversity of rotary ion-translocating ATPases from different organisms and compares the structural, functional, and regulatory features of these enzymes.

Amino Acid Residues ß139, ß189, and ß319 Modulate ADP-Inhibition in Escherichia coli H+-FOF1-ATP Synthase.

Proton-translocating FOF1-ATP synthase (F-type ATPase, F-ATPase or FOF1) performs ATP synthesis/hydrolysis coupled to proton transport across the membrane in mitochondria, chloroplasts, and most eubacteria. The ATPase activity of the enzyme is suppressed in the absence of protonmotive force by several regulatory mechanisms. The most conserved of these mechanisms is noncompetitive inhibition of ATP hydrolysis by the MgADP complex (ADP-inhibition) which has been found in all the enzymes studied. When MgADP binds without phosphate in the catalytic site, the enzyme enters an inactive state, and MgADP gets locked in the catalytic site and does not exchange with the medium. The degree of ADP-inhibition varies in FOF1 enzymes from different organisms. In the Escherichia coli enzyme, ADP-inhibition is relatively weak and, in contrast to other organisms, is enhanced rather than suppressed by phosphate. In this study, we used site-directed mutagenesis to investigate the role of amino acid residues ß139, ß158, ß189, and ß319 of E. coli FOF1-ATP synthase in the mechanism of ADP-inhibition and its modulation by the protonmotive force. The amino acid residues in these positions differ in the enzymes from beta- and gammaproteobacteria (including E. coli) and FOF1-ATP synthases from other eubacteria, mitochondria, and chloroplasts. The ßN158L substitution produced no effect on the enzyme activity, while substitutions ßF139Y, ßF189L, and ßV319T only slightly affected ATP (1 mM) hydrolysis. However, in a mixture of ATP and ADP, the activity of the mutants was less suppressed than that of the wild-type enzyme. In addition, mutations ßF189L and ßV319T weakened the ATPase activity inhibition by phosphate in the presence of ADP. We suggest that residues ß139, ß189, and ß319 are involved in the mechanism of ADP-inhibition and its modulation by phosphate.

ADP-Inhibition of H+-FOF1-ATP Synthase.

H+-FOF1-ATP synthase (F-ATPase, F-type ATPase, FOF1 complex) catalyzes ATP synthesis from ADP and inorganic phosphate in eubacteria, mitochondria, chloroplasts, and some archaea. ATP synthesis is powered by the transmembrane proton transport driven by the proton motive force (PMF) generated by the respiratory or photosynthetic electron transport chains. When the PMF is decreased or absent, ATP synthase catalyzes the reverse reaction, working as an ATP-dependent proton pump. The ATPase activity of the enzyme is regulated by several mechanisms, of which the most conserved is the non-competitive inhibition by the MgADP complex (ADP-inhibition). When ADP binds to the catalytic site without phosphate, the enzyme may undergo conformational changes that lock bound ADP, resulting in enzyme inactivation. PMF can induce release of inhibitory ADP and reactivate ATP synthase; the threshold PMF value required for enzyme reactivation might exceed the PMF for ATP synthesis. Moreover, membrane energization increases the catalytic site affinity to phosphate, thereby reducing the probability of ADP binding without phosphate and preventing enzyme transition to the ADP-inhibited state. Besides phosphate, oxyanions (e.g., sulfite and bicarbonate), alcohols, lauryldimethylamine oxide, and a number of other detergents can weaken ADP-inhibition and increase ATPase activity of the enzyme. In this paper, we review the data on ADP-inhibition of ATP synthases from different organisms and discuss the in vivo role of this phenomenon and its relationship with other regulatory mechanisms, such as ATPase activity inhibition by subunit ε and nucleotide binding in the noncatalytic sites of the enzyme. It should be noted that in Escherichia coli enzyme, ADP-inhibition is relatively weak and rather enhanced than prevented by phosphate.