Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.14 (ATP synthase)
7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

F(1)-ATPase, a water-soluble portion of the enzyme ATP synthase, is a rotary molecular motor driven by ATP hydrolysis. To learn how the kinetics of rotation are regulated, we have investigated the rotational characteristics of a thermophilic F(1)-ATPase over the temperature range 4-50 degrees C by attaching a polystyrene bead (or bead duplex) to the rotor subunit and observing its rotation under a microscope. The apparent rate of ATP binding estimated at low ATP concentrations increased from 1.2 x 10(6) M(-1) s(-1) at 4 degrees C to 4.3 x 10(7) M(-1) s(-1) at 40 degrees C, whereas the torque estimated at 2 mM ATP remained around 40 pN.nm over 4-50 degrees C. The rotation was stepwise at 4 degrees C, even at the saturating ATP concentration of 2 mM, indicating the presence of a hitherto unresolved rate-limiting reaction that occurs at ATP-waiting angles. We also measured the ATP hydrolysis activity in bulk solution at 4-65 degrees C. F(1)-ATPase tends to be inactivated by binding ADP tightly. Both the inactivation and reactivation rates were found to rise sharply with temperature, and above 30 degrees C, equilibrium between the active and inactive forms was reached within 2 s, the majority being inactive. Rapid inactivation at high temperatures is consistent with the physiological role of this enzyme, ATP synthesis, in the thermophile.
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PMID:Temperature dependence of the rotation and hydrolysis activities of F1-ATPase. 1837 15

Chemical crosslinking mediated by short bifunctional reagents has been widely used for determining physical relationships among polypeptides in multisubunit proteins, but less often for functional studies. Here we introduce the approach of tethering polypeptides by using bifunctional reagents containing a lengthy, flexible PEG linker as a form of crosslinking especially suited to functional analyses. The rotary molecular motor ATP synthase was used as a model subject. Single cysteine residues were introduced into selected positions of ATP synthase epsilon subunit, a component of the rotor subcomplex of the enzyme, and the unrelated maltose binding protein (MBP), then the two purified recombinant proteins were crosslinked by means of a dimaleimido-PEG cross-linking agent. Following purification, the epsilon-PEG-MBP was incorporated into membrane-bound ATP synthase by reconstitution with epsilon-depleted F(1)-ATPase and membrane vesicles that had been stripped of endogenous F(1). ATP synthase reconstituted using epsilon-PEG-MBP had reduced ATP hydrolytic activity that was uncoupled from the pumping of H(+), indicating the physical blockage of rotation of the gammaepsilonc(10) rotor by the conjugated MBP, whereas enzyme reconstituted with epsilon-PEG was normal. These results directly demonstrate the feasibility of studying mechanistic features of molecular motors through PEG-based conjugation of unrelated proteins. Since tethering polypeptides provides a means of maintaining proximity without directly specifying or modifying interactions, application of the general method to other types of protein functional studies is envisioned.
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PMID:Tethering polypeptides through bifunctional PEG cross-linking agents to probe protein function: application to ATP synthase. 1844 34

The F(0)F(1)-ATP synthase couples the functions of H(+) transport and ATP synthesis/hydrolysis through the efficient transmission of energy mediated by rotation of the centrally located gamma, epsilon, and c subunits. To understand the gamma subunit role in the catalytic mechanism, we previously determined the partial rate constants and devised a minimal kinetic model for the rotational hydrolytic mode of the F(1)-ATPase enzyme that uniquely fits the pre-steady state and steady state data ( Baylis Scanlon, J. A., Al-Shawi, M. K., Le, N. P., and Nakamoto, R. K. (2007) Biochemistry 46, 8785-8797 ). Here we directly test the model using two single cysteine mutants, betaD380C and betaE381C, which can be used to reversibly inhibit rotation upon formation of a cross-link with the conserved gammaCys-87. In the pre-steady state, the gamma-beta cross-linked enzyme at high Mg.ATP conditions retained the burst of hydrolysis but was not able to release P(i). These data show that the rate-limiting rotation step, k(gamma), occurs after hydrolysis and before P(i) release. This analysis provides additional insights into how the enzyme achieves efficient coupling and implicates the betaGlu-381 residue for proper formation of the rate-limiting transition state involving gamma subunit rotation.
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PMID:A rotor-stator cross-link in the F1-ATPase blocks the rate-limiting step of rotational catalysis. 1862 3

Adenosine triphosphate (ATP), the universal fuel of the cell, is synthesized from adenosine diphosphate (ADP) and inorganic phosphate (P(i)) by 'ATP synthase' (F(O)F(1)-ATPase). During respiration or photosynthesis, an electrochemical potential difference of protons is set up across the respective membranes. This powers the enzyme's electrical rotary nanomotor (F(O)), which drives the chemical nanomotor (F(1)) by elastic mechanical-power transmission, producing ATP with high kinetic efficiency. Attempts to understand in detail the mechanisms of torque generation in this simple and robust system have been both aided and complicated by a wealth of sometimes conflicting data.
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PMID:Torque generation and elastic power transmission in the rotary F(O)F(1)-ATPase. 1945 12

The F(1)F(O) and F(1)-ATPase complexes of Paracoccus denitrificans were isolated for the first time by ion exchange, gel filtration, and density gradient centrifugation into functional native preparations. The liposome-reconstituted holoenzyme preserves its tight coupling between F(1) and F(O) sectors, as evidenced by its high sensitivity to the F(O) inhibitors venturicidin and diciclohexylcarbodiimide. Comparison and N-terminal sequencing of the band profile in SDS-PAGE of the F(1) and F(1)F(O) preparations showed a novel 11-kDa protein in addition to the 5 canonical alpha, beta, gamma, delta, and epsilon subunits present in all known F(1)-ATPase complexes. BN-PAGE followed by 2D-SDS-PAGE confirmed the presence of this 11-kDa protein bound to the native F(1)F(O)-ATP synthase of P. denitrificans, as it was observed after being isolated. The recombinant 11 kDa and epsilon subunits of P. denitrificans were cloned, overexpressed, isolated, and reconstituted in particulate F(1)F(O) and soluble F(1)-ATPase complexes. The 11-kDa protein, but not the epsilon subunit, inhibited the F(1)F(O) and F(1)-ATPase activities of P. denitrificans. The 11-kDa protein was also found in Rhodobacter sphaeroides associated to its native F(1)F(O)-ATPase. Taken together, the data unveil a novel inhibitory mechanism exerted by this 11-kDa protein on the F(1)F(O)-ATPase nanomotor of P. denitrificans and closely related alpha-proteobacteria.
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PMID:A novel 11-kDa inhibitory subunit in the F1FO ATP synthase of Paracoccus denitrificans and related alpha-proteobacteria. 1978 85

The structure of the complex between bovine mitochondrial F(1)-ATPase and a stator subcomplex has been determined at a resolution of 3.2 A. The resolved region of the stator contains residues 122-207 of subunit b; residues 5-25 and 35-57 of F(6); 3 segments of subunit d from residues 30-40, 65-74, and 85-91; and residues 1-146 and 169-189 of the oligomycin sensitivity conferral protein (OSCP). The stator subcomplex represents its membrane distal part, and its structure has been augmented with an earlier structure of a subcomplex containing residues 79-183, 3-123, and 5-70 of subunits b, d, and F(6), respectively, which extends to the surface of the inner membrane of the mitochondrion. The N-terminal domain of the OSCP links the stator with F(1)-ATPase via alpha-helical interactions with the N-terminal region of subunit alpha(E). Its C-terminal domain makes extensive helix-helix interactions with the C-terminal alpha-helix of subunit b from residues 190-207. Subunit b extends as a continuous 160-A long alpha-helix from residue 188 back to residue 79 near to the surface of the inner mitochondrial membrane. This helix appears to be stiffened by other alpha-helices in subunits d and F(6), but the structure can bend inward toward the F(1) domain around residue 146 of subunit b. The linker region between the 2 domains of the OSCP also appears to be flexible, enabling the stator to adjust its shape as it passes over the changing profile of the F(1) domain during a catalytic cycle. The structure of the membrane extrinsic part of bovine ATP synthase is now complete.
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PMID:The structure of the membrane extrinsic region of bovine ATP synthase. 1999 87

Previously melittin, the alpha-helical basic honey bee venom peptide, was shown to inhibit F(1)-ATPase by binding at the beta-subunit DELSEED motif of F(1)F(o)-ATP synthase. Herein, we present the inhibitory effects of the basic alpha-helical amphibian antimicrobial peptides, ascaphin-8, aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, citropin 1.1, dermaseptin, maculatin 1.1, maganin II, MRP, or XT-7, on purified F(1) and membrane bound F(1)F(0)Escherichia coli ATP synthase. We found that the extent of inhibition by amphibian peptides is variable. Whereas MRP-amide inhibited ATPase essentially completely (approximately 96% inhibition), carein 1.8 did not inhibit at all (0% inhibition). Inhibition by other peptides was partial with a range of approximately 13-70%. MRP-amide was also the most potent inhibitor on molar scale (IC(50) approximately 3.25 microM). Presence of an amide group at the c-terminal of peptides was found to be critical in exerting potent inhibition of ATP synthase ( approximately 20-40% additional inhibition). Inhibition was fully reversible and found to be identical in both F(1)F(0) membrane preparations as well as in isolated purified F(1). Interestingly, growth of E. coli was abrogated in the presence of ascaphin-8, aurein 2.2, aurein 2.3, citropin 1.1, dermaseptin, magainin II-amide, MRP, MRP-amide, melittin, or melittin-amide but was unaffected in the presence of carein 1.8, carein 1.9, maculatin 1.1, magainin II, or XT-7. Hence inhibition of F(1)-ATPase and E. coli cell growth by amphibian antimicrobial peptides suggests that their antimicrobial/anticancer properties are in part linked to their actions on ATP synthase.
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PMID:Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides. 2010 May 9

F(1)-ATPase (F(1)), a soluble portion of F(o)F(1)-ATP synthase (F(o)F(1)), is an ATP-driven motor in which gammaepsilon subunits rotate in the alpha(3)beta(3) cylinder. Activity of F(1) and F(o)F(1) from Bacillus PS3 is attenuated by the epsilon subunit in an inhibitory extended form. In this study we observed ATP-dependent transition of epsilon in single F(1) molecules from extended form to hairpin form by fluorescence resonance energy transfer. The results justify the previous bulk experiments and ensure that fraction of F(1) with hairpin epsilon directly determines the fraction of active F(1) at any ATP concentration. Next, mechanical activation and stiffness of epsilon-inhibited F(1) were examined by the forced rotation of magnetic beads attached to gamma. Compared with ADP inhibition, which is another manner of inhibition, rotation by a larger angle was required for the activation from epsilon inhibition when the beads were forced to rotate to ATP hydrolysis direction, and more torque was required to reach the same rotation angle when beads were forced to rotate to ATP synthesis direction. The results imply that if F(o)F(1) is resting in the epsilon-inhibited state, F(o) motor must transmit to gamma a torque larger than expected from thermodynamic equilibrium to initiate ATP synthesis.
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PMID:Activation and stiffness of the inhibited states of F1-ATPase probed by single-molecule manipulation. 2015 86

The F(o)F(1)-ATPase, which synthesizes ATP with a rotary motion, is highly regulated in vivo in order to function efficiently, although there remains a limited understanding of the physiological significance of this regulation. Compared with its bacterial and mitochondrial counterparts, the gamma subunit of cyanobacterial F(1), which makes up the central shaft of the motor enzyme, contains an additional inserted region. Although deletion of this region results in the acceleration of the rate of ATP hydrolysis, the functional significance of the region has not yet been determined. By analysis of rotation, we successfully determined that this region confers the ability to shift frequently into an ADP inhibition state; this is a highly conserved regulatory mechanism which prevents ATP synthase from carrying out the reverse reaction. We believe that the physiological significance of this increased likelihood of shifting into the ADP inhibition state allows the intracellular ATP levels to be maintained, which is especially critical for photosynthetic organisms.
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PMID:Physiological impact of intrinsic ADP inhibition of cyanobacterial FoF1 conferred by the inherent sequence inserted into the gammasubunit. 2042 Nov 99

F(1)-ATPase (F(1)) is the water-soluble portion of ATP synthase and a rotary molecular motor in which the rotary shaft, the gamma subunit, rotates with 120 degrees steps against the alpha(3)beta(3) stator ring upon ATP hydrolysis. While the crystal structures of F(1) exhibit essentially one stable conformational state of F(1), single-molecule rotation studies revealed that there are two stable conformations of F(1) in each 120 degrees step: the ATP-binding dwell state and the catalytic dwell state. This chapter provides the experimental procedure for the determination of which catalytic state the crystal structures of F(1) represent, by the use of a cross-linking technique in the single-molecule rotation assay. The beta and gamma subunits are cross-linked through a disulfide bond between two cysteine residues genetically introduced at the positions where the beta and gamma subunits have a specific contact in the crystal structures of the ADP-bound form. In the single-molecule rotation assay, the cross-linked F(1) shows a pause at the catalytic dwell state that corresponds to the dwell angle in one turn where the beta subunit undergoes ATP hydrolysis. Thus, this experiment reveals not only that the crystal structure represents the catalytic dwell state but also that the ADP-bound beta subunit represents the catalytically active state. A protocol for inhibition of the wild-type F(1) with chemical inhibitors such as adenosine-5'-(beta,gamma-imino)-triphosphate (AMP-PNP) or/and N(3)(-) under crystallization conditions is also provided.
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PMID:Measurement of the conformational state of F(1)-ATPase by single-molecule rotation. 2062 62


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