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Hydrolysis of ATP by the mitochondrial F‐ATPase is inhibited by a protein called IF1. In the parasitic flagellate, Trypanosoma brucei, this protein, known as TbIF1, is expressed exclusively in the procyclic stage, where the F‐ATPase is synthesizing ATP. In the bloodstream stage, where TbIF1 is absent, the F‐ATPase hydrolyzes ATP made by glycolysis and compensates for the absence of a proton pumping respiratory chain by translocating protons into the intermembrane space, thereby maintaining the essential mitochondrial membrane potential. We have defined regions and amino acid residues of TbIF1 that are required for its inhibitory activity by analyzing the binding of several modified recombinant inhibitors to F1‐ATPase isolated from the procyclic stage of T. brucei. Kinetic measurements revealed that the C‐terminal portion of TbIF1 facilitates homodimerization, but it is not required for the inhibitory activity, similar to the bovine and yeast orthologs. However, in contrast to bovine IF1, the inhibitory capacity of the C‐terminally truncated TbIF1 diminishes with decreasing pH, similar to full length TbIF1. This effect does not involve the dimerization of active dimers to form inactive tetramers. Over a wide pH range, the full length and C‐terminally truncated TbIF1 form dimers and monomers, respectively. TbIF1 has no effect on bovine F1‐ATPase, and this difference in the mechanism of regulation of the F‐ATPase between the host and the parasite could be exploited in the design of drugs to combat human and animal African trypanosomiases. The essential ATPase activity of mitochondrial ATP synthase in the pathogenic stage of Trypanosoma brucei is inhibited by a protein inhibitor TbIF1. TbIF1 forms dimers, acts in a pH‐dependent manner, and does not inhibit a mammalian F1‐ATPase. The inhibition of F1‐ATPase by TbIF1 exhibits several parasite‐specific features. Differences between the parasite and host F1‐ATPase inhibition can be exploited in drug design. [ABSTRACT FROM AUTHOR]

Hydrolysis of ATP by the mitochondrial F‐ATPase is inhibited by a protein called IF1. In the parasitic flagellate, Trypanosoma brucei, this protein, known as TbIF1, is expressed exclusively in the procyclic stage, where the F‐ATPase is synthesizing ATP. In the bloodstream stage, where TbIF1 is absent, the F‐ATPase hydrolyzes ATP made by glycolysis and compensates for the absence of a proton pumping respiratory chain by translocating protons into the intermembrane space, thereby maintaining the essential mitochondrial membrane potential. We have defined regions and amino acid residues of TbIF1 that are required for its inhibitory activity by analyzing the binding of several modified recombinant inhibitors to F1‐ATPase isolated from the procyclic stage of T. brucei. Kinetic measurements revealed that the C‐terminal portion of TbIF1 facilitates homodimerization, but it is not required for the inhibitory activity, similar to the bovine and yeast orthologs. However, in contrast to bovine IF1, the inhibitory capacity of the C‐terminally truncated TbIF1 diminishes with decreasing pH, similar to full length TbIF1. This effect does not involve the dimerization of active dimers to form inactive tetramers. Over a wide pH range, the full length and C‐terminally truncated TbIF1 form dimers and monomers, respectively. TbIF1 has no effect on bovine F1‐ATPase, and this difference in the mechanism of regulation of the F‐ATPase between the host and the parasite could be exploited in the design of drugs to combat human and animal African trypanosomiases. The essential ATPase activity of mitochondrial ATP synthase in the pathogenic stage of Trypanosoma brucei is inhibited by a protein inhibitor TbIF1. TbIF1 forms dimers, acts in a pH‐dependent manner, and does not inhibit a mammalian F1‐ATPase. The inhibition of F1‐ATPase by TbIF1 exhibits several parasite‐specific features. Differences between the parasite and host F1‐ATPase inhibition can be exploited in drug design. [ABSTRACT FROM AUTHOR]

The structures of F-ATPases have predominantly been determined from mitochondrial enzymes, and those of the enzymes in eubacteria have been less studied. Paracoccus denitrificans is a member of the α-proteobacteria and is related to the extinct protomitochondrion that became engulfed by the ancestor of eukaryotic cells. The P. denitrificans F-ATPase is an example of a eubacterial F-ATPase that can carry out ATP synthesis only, whereas many others can catalyse both the synthesis and the hydrolysis of ATP. Inhibition of the ATP hydrolytic activity of the P. denitrificans F-ATPase involves the ζ inhibitor protein, an α-helical protein that binds to the catalytic F1 domain of the enzyme. This domain is a complex of three α-subunits and three β-subunits, and one copy of each of the γ-, δ- and ɛ-subunits. Attempts to crystallize the F1-ζ inhibitor complex yielded crystals of a subcomplex of the catalytic domain containing the α- and β-subunits only. Its structure was determined to 2.3 Å resolution and consists of a heterodimer of one α-subunit and one β-subunit. It has no bound nucleotides, and it corresponds to the `open' or `empty' catalytic interface found in other F-ATPases. The main significance of this structure is that it aids in the determination of the structure of the intact membrane-bound F-ATPase, which has been crystallized. [ABSTRACT FROM AUTHOR]

In this article, we discuss single molecule observation of rotational catalysis by E. coli ATP synthase (F-ATPase) using small gold beads. Studies involving a low viscous drag probe showed the stochastic properties of the enzyme in alternating catalytically active and inhibited states. The importance of subunit interaction between the rotor and the stator, and thermodynamics of the catalysis are also discussed. 'Single Molecule Enzymology' is a new trend for understanding enzyme mechanisms in biochemistry and physiology. © 2013 IUBMB Life, 65(3):247-254, 2013 [ABSTRACT FROM AUTHOR]

We discuss the most recent reports on two proton pumps, F-ATPase (ATP synthase) and V-ATPase (endomembrane proton pump). They are formed from similar extrinsic (F 1 or V 1 ) and intrinsic (Fo or Vo) membrane sectors, and couple chemistry and proton transport through subunit rotation for apparently different physiological roles. Emphasis is placed on the stochastic rotational catalysis of F-ATPase and isoforms of V-ATPase. iubmb Life , 58: 318–322, 2006 [ABSTRACT FROM AUTHOR]

Vma5p (subunit C) of the yeast V-ATPase was produced in Escherichia coli and purified to homogeneity. Analysis of secondary structure by circular dichroism spectroscopy showed that Vma5p comprises 64% α-helix and 17% β-sheet content. The molecular mass of this subunit, determined by gel filtration analysis and small angle X-ray scattering (SAXS), was approximately 51 ± 4 kDa, indicating a high hydration level of the protein in solution. The radius of gyration and the maximum size of Vma5p were determined to be 3.74 ± 0.03 and 12.5 ± 0.1 nm, respectively. Using two independent ab initio approaches, the first low-resolution shape of the protein was determined. Vma5p is an elongated boot-shaped particle consisting of two distinct domains. Co-reconstitution of Vma5p to V1 without C from Manduca sexta resulted in a V1–Vma5p hybrid complex and a 20% increase in ATPase hydrolysis activity. [Copyright &y& Elsevier]

Subunit c of the H+ transporting ATP synthase is an essential part of its membrane domain that participates in transmembrane proton conduction. The annular architecture of the subunit c from different species has been previously reported. However, little is known about the type of interactions that affect the formation of c-rings in the ATPase complex. Here we report that subunit c over-expressed in Escherichia coli and purified in non-ionic detergent solutions self-assembles into annular structures in the absence of other subunits of the complex. The results suggest that the ability of subunit c to form rings is determined by its primary structure. [Copyright &y& Elsevier]

1-Hydroxyethane-1,1-diphosphonate (EHDP) and a variety of other diphosphonates, and also pyrophosphate, at millimolar levels were found to inhibit the growth of Streptococcus mutans GS-5. Inhibition appeared to be due mainly to chelation of Mg2+ and could be readily reversed through addition of Mg2+, or less effectively by other divalent cations. The trianionic forms of the diphosphonates or pyrophosphate were more effective inhibitors than the dianionic forms. Diphosphonates and pyrophosphate did not inhibit glycolysis by S. mutans, assayed in terms of glucose utilization, or arginolysis by Streptoccus rattus FA-1, assayed in terms of ammonia production. However, they did act as buffers to moderate pH changes. Diphosphonates also were inhibitors of the F-ATPase of S. mutans by complex mechanisms only partly reversible with divalent cations. They also were inhibitors of the pyrophosphatase of the organism. However, intact cells were impermeable to the compounds, and inhibition of cytoplasmic or membrane enzymes did not appear to be involved in growth inhibition. [ABSTRACT FROM AUTHOR]