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)

To study ESR absorption of mitochondrial ATPase a special flow system was developed allowing to maintain surviving conditions for mitochondria. It has been shown that ESR free radical signal of mitochondria observed at room temperature (g-factor 2.00 and halfwidth of about 15 Gs) depends on their metabolic state. An increase of free radical content during mitochondrial energization can be associated with operation of ATPsynthetase. It is supposed that free radicals take part in the reaction of ADP phosphorylation and that a molecule of ADP itself bound in the active centre of ATPase can become a free radical to facilitate the process of inorganic phosphate incorporation.
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PMID:[Participation of free radicals in ATP synthesis]. 21 18

A spin-labeled photoaffinity ATP analog, 2-N3-2',3'-SL-ATP (2-N3-SL-ATP) was specifically loaded at catalytic (exchangeable) or noncatalytic (nonexchangeable) nucleotide-binding sites on nucleotide-depleted mitochondrial F1-ATPase. Photolysis of the enzyme complexes resulted in the specific modification of beta-Tyr-345 when the catalytic sites were occupied and beta-Tyr-368 when noncatalytic sites were filled. These are the same amino acid assignments that were made previously using 2-N3ATP. The results demonstrate that the attachment of a spin label moiety to the ribose ring does not prevent proper binding of the analog at both types of nucleotide sites on F1-ATPase and suggest that the probe can be used for investigations of the nucleotide-binding sites using ESR spectroscopy. Enzyme that is in complex with the 2-N3-SL-ATP exhibits an ESR spectrum that is typical for highly immobilized nitroxyl radicals both in the dark or after photolysis. Additional peaks in the high- and low-field regions arise due to dipolar spin interactions most likely involving a pair of catalytic and noncatalytic sites. The two sites are calculated to be approximately 15 A apart. This distance, obtained through ESR spectroscopy, combined with the finding that the 2 labeled amino acids are only 23 residues apart from each other, further supports an adenylate kinase-like arrangement of nucleotide binding sites on F1-ATPase where catalytic and noncatalytic sites are in close proximity (Vogel, P. D., and Cross, R. L. (1991) J. Biol. Chem. 266, 6101-6105).
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PMID:Nucleotide binding sites on mitochondrial F1-ATPase. Electron spin resonance spectroscopy and photolabeling by azido-spin-labeled adenine nucleotides support an adenylate kinase-like orientation. 131 7

Under F1-ATPase irradiation by visible light two characteristic features of flavin main activities were detected: I/ESR signal g = 2.00 appearance which was in favor of flavin photochemical electron reduction and 2/photostimulated O2 consumption by F1-ATPase. The dependence of ESR signal g = 2.00 intensity on visible light wavelength completely coincided with the same dependence obtained for proteinless model riboflavin + ADP. Flavin localization in proximity of ADP bound in the enzyme active site was suggested. Under F1-ATPase irradiation by light lambda greater than 350 nm ATP synthesis was obtained similar to the proteinless model riboflavin + ADP + Pinorg. In this model endogenous flavin was suggested to serve as a photosensitizer, its photoexcitement being a model of dark energization of F1-ATPase in oxidative phosphorylation.
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PMID:[Electron paramagnetic resonance of the F1-H+-ATPase complex and adenosine triphosphate synthesis after its irradiation with visible light]. 287 27

The synthesis of an ATP derivative is described, in which a spin-label is attached to the 3'-position of the ribose moiety and an azido group to C8 of the adenine ring (SL-N3-ATP). Irradiation of this compound at 350 nm generates a nitrene, which will react with any functional group in its vicinity. SL-N3-ATP exhibits a strongly immobilized ESR spectrum in a complex with F1-ATPase from beef heart mitochondria. It was covalently incorporated into this enzyme. SL-N3-ATP may be employed in ESR investigations under conditions in which non-covalent interactions are too weak for motionally restricted species to be easily observed.
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PMID:Synthesis of a photoaffinity-spin-labeled derivative of ATP and its first application to F1-ATPase. 289 96

It was shown by ESR technique using flow system combined with ESR-spectrometer that paramagnetic product appearing in the course of oxidative phosphorylation was directly associated with mitochondrial ATPase operation. A decrease of ESR signal intensity and the changes of its form observed on mitochondria uncoupled by 2,4-dinitrophenol as compared with those inhibited by olygomicin suggest that in the case of olygomycin block a free radical ATPase linked intermediate can be recorded, the ESR signal of which seems to be partly due to flavin semiquinone of ATPsynthetase itself.
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PMID:[Detection of a paramagnetic product developing during oxidative phosphorylation in mitochondria]. 625 23

ATP synthase (F0F1) is driven by an electrochemical potential of H+ (delta microH+). F0F1 is composed of an ion-conducting portion (F0) and a catalytic portion (F1). The subunit composition of F1 is a alpha 3 beta 3 gamma delta epsilon. The active alpha 3 beta 3 oligomer, characterized by X-ray crystallography, has been obtained only from thermophilic F1 (TF1). We proposed in 1984 that ATP is released from the catalytic site (C site) by a conformational change induced by the beta DELSEED sequence via gamma delta epsilon-F0. In fact, cross-linking of beta DELSEED to gamma stopped the ATP-driven rotation of gamma in the center of alpha 3 beta 3. The torque of the rotation is estimated to be 420 pN x A from the delta microH+ and H(+)-current through F0F1. The angular velocity (omega) of gamma is the rate-limiting step, because delta microH+ increased the Vmax of H+ current through F0, but not the Km(ATP). The rotational unit of F0 (= ab2c10) is pi/5, while that in alpha 3 beta 3 is 2 pi/3. This difference is overcome by an analog-digital conversion via elasticity around beta DELSEED with a threshold to release ATP. The alpha beta distance at the C site is about 9.6 A (2,8-diN3-ATP), and tight Mg-ATP binding in alpha 3 beta 3 gamma was shown by ESR. The rotational relaxation of TF1 is too rapid (phi = 100 nsec), but the rate of AT(D)P-induced conformational change of alpha 3 beta 3 measured with a synchrotron is close to omega. The ATP bound between the P-loop and beta E188 is released by the shift of beta DELSEED from gamma RGL. Considering the viscosity resistance and inertia of the free rotor (gamma-c), there may be a stator containing OSCP (= delta of TF1) and F0-d to hold free rotation of alpha 3 beta 3.
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PMID:The energy transmission in ATP synthase: from the gamma-c rotor to the alpha 3 beta 3 oligomer fixed by OSCP-b stator via the beta DELSEED sequence. 895 Oct 89

The isolation of ATP synthase (F0F1) (82) and F0 (83) 34 years ago finally revealed that F0F1 is a motor composed of F0 (ion-motor, abc subunits) and F1 (ATP-motor, alpha 3 beta 3 gamma delta epsilon subunits) (Fig. 1). The single molecule videotape (4, 5, 65, 66) revealed that gamma epsilon axis of F1 rotates counterclockwise, proceeds by each 2 pi/3 step, and is driven by torque of 42 pN.nm (12) with nearly 100% efficiency (5) (Fig. 4). The motor is composed of a rotor (gamma epsilon-F0-c) and a stator (alpha 3 beta 3 delta-F0-ab), and the rotor is connected to a shaft (gamma epsilon). Since F0F1 is driven by delta microH+ (9, 10, 84), biophysical studies on stable TF0F1 (1, 7) are essential to elucidate the mechanism. These include nanomechanics (4, 5) (Fig. 4), crystallography (2, 3) (Figs. 2 and 3), NMR (51, 52), ESR (56), synchrotron analysis (3, 28), and electrophysiology (10, 25). The KmATP value of rotation is 0.8 microM, with the Vmax of 3.9 rps (5). This corresponds to the bi-site catalysis in proton transport by F0F1 (10, 70, 84). X-ray crystallography of MF1 (2) and the alpha 3 beta 3 oligomer of TF1 (3) (Fig. 2) together with mutation analyses revealed the role of residues in the rotation. The idea of elastic energy store is proposed in alpha 3 beta 3 gamma during the stepping time (up to a few sec) after the ATP binding. Biological studies have partially clarified the genetic and kinetic regulation of the rotation in MF1. Both theories (6, 7, 62, 64, 85) and the biological significance (17) of the intramolecular rotation of F0F1 await further studies, especially those of F0 and minor subunits.
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PMID:Biophysical studies on ATP synthase. 1046 71

A variety of different approaches has been used during the last couple of decades to investigate structure and function relationships within the catalytic portion of the F0F1-ATP synthase and of its interactions with the proton-translocator F0. In our group, we employ ESR spectroscopy with the use of stable organic radicals, so-called spin labels, as reporter groups. The radicals are either attached to substrates/ligands or specifically inserted into the protein structure by "site-specific spin labeling." Both approaches bear intrinsic advantages for their special uses and result in the specific information that is available through ESR, e.g., structural changes due to binding of effector molecules (e.g., Mg2+ ions), conformational transitions during catalytic turnover, distance information on radicals bound at 20 A or less, and information on the binding characteristics of labeled substrates. This review summarizes the results of a variety of different approaches we have used during the last years to study, with the help of ESR spectroscopy, the structure of the nucleotide binding sites of F1-ATPases of different origins as well as interactions with F0 subunits.
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PMID:Insights into ATP synthase structure and function using affinity and site-specific spin labeling. 1176 3

The photoaffinity spin-labeled ATP analog, 2-N3-SL-adenosine triphosphate (ATP), was used to covalently modify isolated beta-subunits from F1-ATPase of the thermophilic bacterium PS3. Approximately 1.2 mol of the nucleotide analog bound to the isolated subunit in the dark. Irradiation leads to covalent incorporation of the nucleotide into the binding site. ESR spectra of the complex show a signal that is typical for protein-immobilized radicals. Addition of isolated alpha-subunits to the modified beta-subunits results in ESR spectra with two new signals indicative of two distinctly different environments of the spin-label, e.g., two distinctly different conformations of the catalytic sites. The relative ratio of the signals is approx 2:1 in favor of the more closed conformation. The data show for the first time that when nucleotides are bound to isolated beta-subunits, binding of alpha-subunits induces asymmetry in the catalytic sites even in the absence of the gamma-subunit.
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PMID:Association of alpha-subunits with nucleotide-modified beta-subunits induces asymmetry in the catalytic sites of the F1-ATPase alpha3beta3-hexamer. 1471 74

The structure of the external stalk and its function in the catalytic mechanism of the F(0)F(1)-ATP synthase remains one of the important questions in bioenergetics. The external stalk has been proposed to be either a rigid stator that binds F(1) or an elastic structural element that transmits energy from the small rotational steps of subunits c to the F(1) sector during catalysis. We employed proteomics, sequence-based structure prediction, molecular modeling, and electron spin resonance spectroscopy using site-directed spin labeling to understand the structure and interfacial packing of the Escherichia coli b-subunit homodimer external stalk. Comparisons of bacterial, cyanobacterial, and plant b-subunits demonstrated little sequence similarity. Supersecondary structure predictions, however, show that all compared b-sequences have extensive heptad repeats, suggesting that the proteins all are capable of packing as left-handed coiled-coils. Molecular modeling subsequently indicated that b(2) from the E. coli ATP synthase could pack into stable left-handed coiled-coils. Thirty-eight substitutions to cysteine in soluble b-constructs allowed the introduction of spin labels and the determination of intersubunit distances by ESR. These distances correlated well with molecular modeling results and strongly suggest that the E. coli subunit b-dimer can stably exist as a left-handed coiled-coil.
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PMID:Structure of the cytosolic part of the subunit b-dimer of Escherichia coli F0F1-ATP synthase. 1832 47


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