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)

Signal assignment and secondary structural analysis of uniformly [13C, 15N] labeled H+-ATP synthase subunit c from E. coli (79 residues) in the solid state were carried out by two- and three-dimensional solid-state NMR under magic-angle spinning. The protein took on a unique structure even in the solid state from the 13C linewidths of about 1.7 ppm. On the basis of several inter- and intra-residue 13C-13C and 13C-15N chemical shift correlations, 78% of Calpha, 72% of Cbeta, 62% of C' and 61% of NH signals were assigned, which provided the secondary structure information for 84% of the 79 residues. Here, inter-residue correlations involving Gly, Ala, Pro and side-chains and a higher resolution in the 3D spectrum were significantly useful for the sequence specific assignment. On top of this, the 13C-13C correlation spectra of subunit c was analyzed by reproducing experimental cross peaks quantitatively with chemical shift prediction and signal-intensity calculation based on the structure. It revealed that the subunit c in the solid state could be specified by alpha-helices with a loop structure in the middle (at sequence 41-45) as in the case of the solution structure in spite of additional extended conformations at 76-79 at the C-terminus.
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PMID:Signal assignment and secondary structure analysis of a uniformly [13C, 15N]-labeled membrane protein, H +-ATP synthase subunit c, by magic-angle spinning solid-state NMR. 1708 Feb 95

Two highly conserved amino acid residues near the C-terminus within the gamma subunit of the mitochondrial ATP synthase form a "catch" with an anionic loop on one of the three beta subunits within the catalytic alphabeta hexamer of the F1 segment [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. Forming the catch is considered to be an essential step in cooperative nucleotide binding leading to gamma subunit rotation. The analogous residues, Arg304 and Gln305, in the chloroplast F1 gamma subunit were changed to leucine and alanine, respectively. Each mutant gamma was assembled together with alpha and beta subunits from Rhodospirillum rubrum F1 into a hybrid photosynthetic F1 that carries out both MgATPase and CaATPase activities and ATP-dependent gamma rotation [Tucker, W. C., Schwarcz, A., Levine, T., Du, Z., Gromet-Elhanan, Z., Richter, M. L. and Haran, G. (2004) J. Biol. Chem. 279, 47415-47418]. Surprisingly, changing Arg304 to leucine resulted in a more than 2-fold increase in the kcat for MgATP hydrolysis. In contrast, changing Gln305 to alanine had little effect on the kcat but completely abolished the well-known stimulatory effect of the oxyanion sulfite on MgATP hydrolysis. The MgATPase activities of combined mutants with both residues substituted were strongly inhibited, whereas the CaATPase activities were inhibited, but to a lesser extent. The results indicate that the C-terminus of the photosynthetic F1 gamma subunit, like its mitochondrial counterpart, forms a catch with the alpha and beta subunits that modulates the nucleotide binding properties of the catalytic site(s). The catch is likely to be part of an activation mechanism, overcoming inhibition by free mg2+ ions, but is not essential for cooperative nucleotide exchange.
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PMID:Mutations within the C-terminus of the gamma subunit of the photosynthetic F1-ATPase activate MgATP hydrolysis and attenuate the stimulatory oxyanion effect. 1728 58

A complex of gamma, epsilon, and c subunits rotates in ATP synthase (FoF(1)) coupled with proton transport. A gold bead connected to the gamma subunit of the Escherichia coli F(1) sector exhibited stochastic rotation, confirming a previous study (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126-4131). A similar approach was taken for mutations in the beta subunit key region; consistent with its bulk phase ATPase activities, F(1) with the Ser-174 to Phe substitution (betaS174F) exhibited a slower single revolution time (time required for 360 degree revolution) and paused almost 10 times longer than the wild type at one of the three 120 degrees positions during the stepped revolution. The pause positions were probably not at the "ATP waiting" dwell but at the "ATP hydrolysis/product release" dwell, since the ATP concentration used for the assay was approximately 30-fold higher than the K(m) value for ATP. A betaGly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of betaS174F. The revertant (betaG149A/betaS174F) exhibited similar rotation to the wild type, except that it showed long pauses less frequently. Essentially the same results were obtained with the Ser-174 to Leu substitution and the corresponding revertant betaG149A/betaS174L. These results indicate that the domain between beta-sheet 4 (betaSer-174) and P-loop (betaGly-149) is important to drive rotation.
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PMID:Rotational catalysis of Escherichia coli ATP synthase F1 sector. Stochastic fluctuation and a key domain of the beta subunit. 1751 93

A complex of gamma, epsilon, and c subunits rotates in ATP synthase (F(o)F(1)) coupling with proton transport. Replacement of betaSer174 by Phe in beta-sheet4 of the beta subunit (betaS174F) caused slow gamma subunit revolution of the F(1) sector, consistent with the decreased ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F1 sector. Stochastic fluctuation and a key domain of the beta subunit, J. Biol. Chem. 282 (2007) 20698-20704]. Modeling of the domain including beta-sheet4 and alpha-helixB predicted that the mutant betaPhe174 residue undergoes strong and weak hydrophobic interactions with betaIle163 and betaIle166, respectively. Supporting this prediction, the replacement of betaIle163 in alpha-helixB by Ala partially suppressed the betaS174F mutation: in the double mutant, the revolution speed and ATPase activity recovered to about half of the levels in the wild-type. Replacement of betaIle166 by Ala lowered the revolution speed and ATPase activity to the same levels as in betaS174F. Consistent with the weak hydrophobic interaction, betaIle166 to Ala mutation did not suppress betaS174F. Importance of the hinge domain [phosphate-binding loop (P-loop)/alpha-helixB/loop/beta-sheet4, betaPhe148-betaGly186] as to driving rotational catalysis is discussed.
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PMID:Effects of mutations in the beta subunit hinge domain on ATP synthase F1 sector rotation: interaction between Ser 174 and Ile 163. 1798 92

Two highly conserved amino acid residues, an arginine and a glutamine, located near the C-terminal end of the gamma subunit, form a "catch" by hydrogen bonding with residues in an anionic loop on one of the three catalytic beta subunits of the bovine mitochondrial F1-ATPase [Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. The catch is considered to play a critical role in the binding change mechanism whereby binding of ATP to one catalytic site releases the catch and induces a partial rotation of the gamma subunit. This role is supported by the observation that mutation of the equivalent arginine and glutamine residues in the Escherichia coli F1 gamma subunit drastically reduced all ATP-dependent catalytic activities of the enzyme [Greene, M. D., and Frasch, W. D. (2003) J. Biol. Chem. 278, 5194-5198]. In this study, we show that simultaneous substitution of the equivalent residues in the chloroplast F1 gamma subunit, arginine 304 and glutamine 305, with alanine decreased the level of proton-coupled ATP synthesis by more than 80%. Both the Mg2+-dependent and Ca2+-dependent ATP hydrolysis activities increased by more than 3-fold as a result of these mutations; however, the sulfite-stimulated activity decreased by more than 60%. The Mg2+-dependent, but not the Ca2+-dependent, ATPase activity of the double mutant was insensitive to inhibition by the phytotoxic inhibitor tentoxin, indicating selective loss of catalytic cooperativity in the presence of Mg2+ ions. The results indicate that the catch residues are required for efficient proton coupling and for activation of multisite catalysis when MgATP is the substrate. The catch is not, however, required for CaATP-driven multisite catalysis or, therefore, for rotation of the gamma subunit.
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PMID:C-Terminal mutations in the chloroplast ATP synthase gamma subunit impair ATP synthesis and stimulate ATP hydrolysis. 1809 10

The procyclic form of Trypanosoma brucei is a parasitic protozoan that normally dwells in the midgut of its insect vector. In vitro, this parasite prefers d-glucose to l -proline as a carbon source, although this amino acid is the main carbon source available in its natural habitat. Here, we investigated how l -proline is metabolized in glucose-rich and glucose-depleted conditions. Analysis of the excreted end products of (13)C-enriched l -proline metabolism showed that the amino acid is converted into succinate or l -alanine depending on the presence or absence of d-glucose, respectively. The fact that the pathway of l -proline metabolism was truncated in glucose-rich conditions was confirmed by the analysis of 13 separate RNA interference-harboring or knock-out cell lines affecting different steps of this pathway. For instance, RNA interference studies revealed the loss of succinate dehydrogenase activity to be conditionally lethal only in the absence of d-glucose, confirming that in glucose-depleted conditions, l -proline needs to be converted beyond succinate. In addition, depletion of the F(0)/F(1)-ATP synthase activity by RNA interference led to cell death in glucose-depleted medium, but not in glucose-rich medium. This implies that, in the presence of d-glucose, the importance of the F(0)/F(1)-ATP synthase is diminished and ATP is produced by substrate level phosphorylation. We conclude that trypanosomes develop an elaborate adaptation of their energy production pathways in response to carbon source availability.
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PMID:Glucose-induced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei. 1843 Jul 32

The rotation of F1-ATPase (F1) is driven by the open/close bending motion of the beta subunit. The mechanism underlying the bending motion was investigated for the F1beta monomer from thermophilic Bacillus PS3 (TF1beta) in solution, using mutagenesis and NMR. The hydrogen bond networks involving the side chains of Lys-164 (numbering for TF1beta; 162 for mitochondrial F1beta in parentheses), Thr-165(163), Arg-191(189), Asp-252(256), Asp-311(315), and Arg-333(337) in the catalytic region are significantly different for the ligand-bound and freebeta subunits in the crystal structures of mitochondrial F1. The role of each amino acid residue was examined by Ala substitution. beta(K164A) reduced the affinity constant for 5'-adenyl-beta,gamma-imidodiphosphate by 20-fold and abolished the conformational change associated with nucleotide binding and the ATPase activity of alpha3beta(K164A)3gamma.beta(T165A) and beta(D252A) exhibited no effect on the binding affinity but abolished the conformational change and the ATPase activity. The chemical shift perturbation of backbone amide signals of the segmentally labeled beta(mutant)s indicated stepwise propagation of the open/close conversion on ligand binding. The key action in the conversion is the switching of the hydrogen-bonding partner of Asp-252 from Lys-164 to Thr-165. Residual dipolar coupling analysis revealed that the closed conformation of the beta monomer was more closed than that in the crystal structure and was different for MgATP- and MgADP-bound beta subunits. Actually, MgATP induced a conformational change around Tyr-307 (311 for MF1beta), whereas MgADP did not. The significance of these findings is discussed in connection with the catalytic rotation of F1-ATPase.
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PMID:Stepwise propagation of the ATP-induced conformational change of the F1-ATPase beta subunit revealed by NMR. 1902 77

The membrane-embedded rotor in the F(0) sector of proton-translocating ATP synthases is formed from hairpin-like c-subunits that are protonated and deprotonated during energization of ATP synthesis. This study focuses on two c-subunit motifs that are unique to synthases of extremely alkaliphilic Bacillus species. One motif is the AXAXAXA sequence found in the N-terminal helix-1 instead of the GXGXGXG of non-alkaliphiles. Quadruple A-->G chromosomal mutants of alkaliphilic Bacillus pseudofirmus OF4 retain 50% of the wild-type hydrolytic activity (ATPase) but <18% of the ATP synthase capacity at high pH. Consistent with a structural impact of the four alanine replacements, the mutant ATPase activity showed enhanced inhibition by dicyclohexylcarbodiimide, which blocks the helix-2 carboxylate. Single, double, or triple A-->G mutants exhibited more modest defects, as monitored by malate growth. The key carboxylate is in the second motif, which is P(51)XXE(54)XXP in extreme alkaliphiles instead of the (A/G)XX(E/D)XXP found elsewhere. Mutation of Pro(51) to alanine had been shown to severely reduce malate growth and ATP synthesis at high pH. Here, two Pro(51) to glycine mutants of different severities retained ATP synthase capacity but exhibited growth deficits and proton leakiness. A Glu(54) to Asp(54) change increased proton leakiness and reduced malate growth 79-90%. The Pro(51) and the Glu(54) mutants were both more dicyclohexylcarbodiimide-sensitive than wild type. The results highlight the requirement for c-subunit adaptations to achieve alkaliphile ATP synthesis with minimal cytoplasmic proton loss and suggest partial suppression of some mutations by changes outside the atp operon.
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PMID:Characterization of the Functionally Critical AXAXAXA and PXXEXXP Motifs of the ATP Synthase c-Subunit from an Alkaliphilic Bacillus. 1917 24

Previous work has shown that the essential R210 of subunit a in the Escherichia coli ATP synthase can be switched with a conserved glutamine Q252 with retention of a moderate level of function, that a third mutation P204T enhances this function, and that the arginine Q252R can be replaced by lysine without total loss of activity. In this study, the roles of P204T and R210Q were examined. It was concluded that the threonine in P204T is not directly involved in function since its replacement by alanine did not significantly affect growth properties. Similarly, it was concluded that the glutamine in R210Q is not directly involved with function since replacement by glycine results in significantly enhanced function. Not only did the rate of ATP-driven proton translocation increase, but also the sensitivity of ATP hydrolysis to inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) rose to more than 50%. Finally, mutations at position E219, a residue near the proton pathway, were used to test whether the Arginine-switched mutant uses the normal proton pathway. In a wild type background, the E219K mutant was confirmed to have greater function than the E219Q mutant, as has been shown previously. This same unusual result was observed in the triple mutant background, P204T/R210Q/Q252R, suggesting that the Arginine-switched mutants are using the normal proton pathway from the periplasm.
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PMID:A more robust version of the Arginine 210-switched mutant in subunit a of the Escherichia coli ATP synthase. 1936 69

The sustained elevation of plasma and interstitial catecholamine levels, namely adrenaline (ADR), and the generation of reactive oxygen species (ROS) are well recognized hallmarks of several cardiopathologic conditions, like cardiac ischemia/reperfusion (I/R) and heart failure (HF). The present work aimed to investigate the proteomics and energetic metabolism of cardiomyocytes incubated with ADR and/or ROS. To mimic pathologic conditions, freshly isolated calcium-tolerant cardiomyocytes from adult rat were incubated with ADR alone or in the presence of a system capable of generating ROS [(xanthine with xanthine oxidase) (XXO)]. Two-dimensional electrophoresis with matrix-assisted laser desorption/ionization and time-of-flight mass spectrometer analysis were used to define protein spot alterations in the cardiomyocytes incubated with ADR and/or ROS. Moreover, the energetic metabolism and the activity of mitochondrial complexes were evaluated by nuclear magnetic resonance and spectrophotometric determinations, respectively. The protein extract was mainly constituted by cardiac mitochondrial proteins and the alterations found were included in five functional classes: (i) structural proteins, notably myosin light chain-2; (ii) redox regulation proteins, in particular superoxide dismutase (SOD); (iii) energetic metabolism proteins, encompassing ATP synthase alpha chain and dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex; (iv) stress response proteins, like the heat shock proteins; and (v) regulatory proteins, like cytochrome c and voltage-dependent anion channel 1. The XXO system elicited alterations in cardiac contractile proteins, as they showed high levels of cleavage, and also altered energetic metabolism, through increased lactate and alanine levels. The cardiomyocytes incubation with ADR resulted in an accentuated increase in mitochondrial complexes activity and the decrease in alanine/lactate ratio, thus reflecting a high cytosolic NADH/NAD(+) ratio. Furthermore, an increase in manganese SOD expression and total SOD activity occurred in the ADR group, as the increase in the mitochondrial complexes presumably led to higher 'electron leakage'. The modifications in proteins, enzymes activity, and energetic metabolism were indicative that different pathways are activated by catecholamines and ROS. These alterations altogether determine the I/R and HF specific features and contribute for the initiation or aggravation of those cardiopathologic conditions.
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PMID:Adrenaline and reactive oxygen species elicit proteome and energetic metabolism modifications in freshly isolated rat cardiomyocytes. 1946 73


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