Fi-ATPase

coli Fi -ATPase consists of the structure aifijydc. The a-subunit and /3-subunit each independently possesses a nucleotide-binding site and the catalytic site is believed to be on the /3-subunit or at the interface between the a- and /3-subunits (for reviews, see Ref. 59 and Chapter 11 of this volume). E. coli Fi -ATPase was more effectively inactivated by AP3-PL and AP4-PL than AP2-PL. The binding of one mole of AP3-PL to one mole of Fi resulted in an almost complete loss of activity 70% of the label bound to Fi to the a-subunit and the rest to the /3-subunit. The sequence analysis of the Fi modified by AP3-PL in the absence of Mg2+ revealed that Lys-201 of the a-subunit and Lys-155 of the /3-subunit are the 

Mg2+ is essential for the hydrolysis of ATP by F. Addition of Mg2+ decreased the concentration of AP3-PL required for half-maximum inactivation (10 pM and 2.5 pM in the absence and presence of Mg2+, respectively) and changed the distribution pattern of bound AP3-PL without change in inactivation stoichiometry.61) In the presence of Mg2+, the /3-subunit was almost exclusively labeled by the reagent. Sequence study identified Lys-155 and Lys-201 of the /3-subunit as the labeled sites, suggesting that this residue as well as Lys-155 of the /8-subunit and Lys-201 of the a-subunit are present at the catalytic site. 

Lys-155 of the /3-subunit is in the consensus sequence for the nucleotide-binding (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, where the conserved residues are underlined). The results of site-directed mutagenesis studies of this sequence suggest its importance for ATP hydrolysis.62-64) 

Modification of Bovine Mitochondrial Fi-ATPase by FSBA65 67) and Fluoro-sulfonylbenzoyl Inosine (FSBI)68  

The inactivation of bovine Fi -ATPase by FSBA exhibited biphasic kinetics. A double reciprocal plot of the inactivation rate for the fast phase against FSBA concentration gave a curved line, whereas the same type of plot for the slow phase yielded a straight line, giving a Ka value of 0.23 mM. The slow phase was diminished when the enzyme was inactivated in the presence of 0.2 M phosphate. On complete inactivation, about three moles of FSBA bound to one mole of bovine Fi -ATPase. Regardless of the presence of phosphate, His-427 of the (8-subunit was dominantly modified at pH 6, whereas Tyr-368 of the /8-subunit was dominantly modified at pH 8. At pH 7, the two residues were modified in a similar ratio. 

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A sequence of ten amino acids (ICS-D-KTGTLT) around the phosphorylation site of Na,K-ATPase (Asp ) is highly conserved among the Na,K-, H,K-, Ca-, and Id-pumps [6]. There is also homology with the subunit of FpATP synthetase of mitochondria and chloroplasts (see [6]) except that Asp is replaced by Thr. Accordingly a covalent phosphorylated intermediate is not formed in Fi-ATPase. Mutagenesis of the phosphorylated aspartate residue in Na,K-ATPase [82], Ca-ATPase [87], or H-ATPase [88] completely blocks activity. 

Between 1955 and 1960 various sub-mitochondrial preparations were developed to give vesicles comprising only sealed inner mitochondrial membranes. Cooper and Lehninger used digitonin extraction Lardy and Kielley Bronk prepared sub-mitochondrial particles by sonication. At this time, too, Racker and his colleagues isolated Fq/F1 particles from mitochondria and showed that a separated FI particle behaved as an ATPase. The F0 portion had no enzymic properties but conferred oligomycin sensitivity on the FI ATPase. The orientation of these sub-mitochondrial vesicles (inside-out or vice-versa) was shown by the position in electron micrographs of the dense (FI) particles which in normal intact mitochondria project into the matrix and so define the surface of the inner mitochondrial membrane. 

F -ATPase Driven Nanomotors. Another type of biological driven engine is that of Fi-adenosine triphosphate synthease (Fi-ATPase) which hydrolyzes the ATP in the surrounding medium. Kinosita Jr. et al. observed the rotation of an actin filament attached to the Fi - ATPase motor. Later, Montemagno followed with the incorporation of a nickel nanorod with the Fi-ATPase motor. The outcome was the rotation of the... 

H. Noji, R. Yasuda, M. Yoshia, andK. Kinosita Jr., Direct observation of the rotation of Fi-ATPase, Nature 386, 299-302 (1997). 

Ivey DM, Ljungdahl LG. 1986. Purification and characterization of the Fi-ATPase from Clostridium thermoaceticum. J Bacteriol 165 252-7. 

The Fi ATPase consists of a cluster of at least five different proteins which are linked to another complex of proteins within the inner membrane, known as Fq. The combination of the F, and Fo protein complexes is Complex V (Table 9.4). Simple diagrams to illustrate their structural relationship and their function in the generation of ATP are presented in Figures 9.11(a) and (b). 

The concept of rotational catalysis by ATP synthase is based on (a) P and 0 exchange rate data attesting to strong cooperativity with sequential participation of several catalytic sites (b) Pi and ATP 0-isotopomer distributions indicating that all catalytic sites exhibit identical catalysis and (c) that catalysis is strongly influenced by the y-subunit whose primary structure was not likely to account for spatially similar interactions with the /3-subunits . The model was found to be compatible with the 2.8 A resolution structure of bovine heart mitochondrial Fi-ATPase. ... 

A highly reactive compound containing a neutral divalent carbon with two nonbondmg electrons (ie.,. CR2 or a substitution derivative). The nonbonding electrons can have parallel spins (triplet state) or antiparallel spins (singlet state). The parent species, iCRz, is also known as methylene. A number of carbene derivatives have been used as photoaffinity labels of proteins. Irradiation of 3 -0-(4-benzoyl)benzoyl-ATP will cause 70% inactivation of mitochondrial Fi-ATPase. ... 

Because of the irreversibility of the catalysis steps, once ATP is bound to the Fi-ATPase it has to be hydrolyzed to ADP and P, and subsequently release the products from the catalytic site. As E.ADP.P, is an intermediate in ATP hydrolysis, quasi-steady state considerations imply that the rate of formation of E.ADP.Pj is equal to its rate of consumption, i.e. ... 

The structure of Fi-ATPase from bovine heart mitochondria determined at 2.8 A resolution. Nature 370, 621-628. 

The 2.80 A structure of rat liver Fi-ATPase configuration of a critical intermediate in ATP synthesis-hydrolysis. Proc. Natl. Acad. Sci. USA 95, 11,065-11,070. 

Cabezon, E., Montgomery, M.G., Leslie, A.G.W., Walker, J.E. (2003) The structure of bovine Fi-ATPase in complex with its regulatory protein IFi Nat. Struct. Biol 10, 744-750. 

Weber, J. Senior, A.E. (1997) Catalytic mechanism of Fi-ATPase. Biochim. Biophys. Acta 1319, 19-58. 

Yasuda, R., Nojl, H., Kinosita, K., Jr., Yoshida, M. (1998) Fi-ATPase is a highly efficient molecular motor that rotates with discrete 120° steps. Cell 93, 1117-1124. 

Figure 17.16 Schematic representation of a nanomechanical device powered by the Fi-ATPase molecular motor.145 (Adapted with permission from V. Balzani et al., ChemPhysChem 2008, 9, 202-220. Copyright Wiley-VCH Verlag GmbH Co. KGaA.)...

Fig. 11.5 Uni-site (single-site) and multi-site (steady-state) catalysis by Fi-ATPase.

Fig. 11.7 The glycine-rich sequences of adenylate kinase, ras protein, and the fi subunit of Fi-ATPase.

Elucidation of the crystal structure of the bovine heart mitochondrial Fi-ATPase (Abraham et al., 1994, Gibbons et. al., 2000) focused attention on rotational catalysis in coupling ATP synthesis and hydrolysis with the proton translocation. Electron microscopy and X-ray structural analysis studies have shown that theFi(part of the enzyme is separated from the Fo by a narrow stalk of around 45 A. 

DNAPa, DNAPI (DNAL, FI ATPase, HIV-1 RT, IKK, iNOS, 5-LOX, NADH DH, Na+, K+-ATPase, NEP, PK, 5aR, succinate DH, TOPII, TPO) [antibacterial, antigonadotropic, apoptotic]... 

Solanum ATPase inhibitor protein Solanum tuberosum (Solanaceae) [tuber mitochondria] Fi-ATPase (potato yeast)... 

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