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)

The functional role of essential residue alpha-Arg-376 in the catalytic site of F1-ATPase was studied. The mutants alpha R376C, alpha R376Q, and alpha R376K were constructed, and combined with the mutation beta Y331W, to investigate catalytic site nucleotide-binding parameters, and to assess catalytic transition state formation by measurement of MgADP-fluoroaluminate binding. Each mutation caused large impairment of ATP synthesis and hydrolysis. Despite the apparent proximity of alpha-Arg-376 to bound nucleoside di- and triphosphate in published X-ray structures, the mutations had little effect on MgADP or MgATP binding affinities, particularly at the highest affinity catalytic site, site 1. Both Cys and Gln mutants abolished transition state formation, demonstrating that alpha-Arg-376 is normally involved at this step of catalysis. A model of the F1-ATPase catalytic transition state structure is presented and discussed. The Lys mutant, although severely impaired, supported transition state formation, suggesting that an additional essential role for the alpha-Arg-376 guanidinium group exists, likely in alpha/beta conformational signal transmission required for steady-state catalysis. Parallels between alpha-Arg-376 and GAP/G-protein "arginine finger" residues are evident.
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PMID:Importance of F1-ATPase residue alpha-Arg-376 for catalytic transition state stabilization. 1056 31

In the crystal structure of the mitochondrial F(1)-ATPase, the beta-Thr(163) residue was identified as a ligand to Mg(2+) and the beta-Glu(188) as directly involved in catalysis. We replaced the equivalent beta-Thr(159) of the chromatophore F(0)F(1) ATP synthase of Rhodospirillum rubrum with Ser, Ala, or Val and the Glu(184) with Gln or Lys. The mutant beta subunits were isolated and tested for their capacity to assemble into a beta-less chromatophore F(0)F(1) and restore its lost activities. All of them were found to bind into the beta-less enzyme with the same efficiency as the wild type beta subunit, but only the beta-Thr(159) --> Ser mutant restored the activity of the assembled enzyme. These results indicate that both Thr(159) and Glu(184) are not required for assembly and that Glu(184) is indeed essential for all the membrane-bound chromatophore F(0)F(1) activities. A detailed comparison between the wild type and the beta-Thr(159) --> Ser mutant revealed a rather surprising difference. Although this mutant restored the wild type levels and all specific properties of this F(0)F(1) proton-coupled ATP synthesis as well as Mg- and Mn-dependent ATP hydrolysis, it did not restore at all the proton-decoupled CaATPase activity. This clear difference between the ligands for Mg(2+) and Mn(2+), where threonine can be replaced by serine, and Ca(2+), where only threonine is active, suggests that the beta-subunit catalytic site has different conformational states when occupied by Ca(2+) as compared with Mg(2+). These different states might result in different interactions between the beta and gamma subunits, which are involved in linking F(1) catalysis with F(0) proton-translocation and can thus explain the complete absence of Ca-dependent proton-coupled F(0)F(1) catalytic activity.
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PMID:Mutations in the beta-subunit Thr(159) and Glu(184) of the Rhodospirillum rubrum F(0)F(1) ATP synthase reveal differences in ligands for the coupled Mg(2+)- and decoupled Ca(2+)-dependent F(0)F(1) activities. 1062 25

The three catalytic sites of the F(O)F(1) ATP synthase interact through a cooperative mechanism that is required for the promotion of catalysis. Replacement of the conserved alpha subunit Arg-376 in the Escherichia coli F(1) catalytic site with Ala or Lys resulted in turnover rates of ATP hydrolysis that were 2 x 10(3)-fold lower than that of the wild type. Mutant enzymes catalyzed hydrolysis at a single site with kinetics similar to that of the wild type; however, addition of excess ATP did not chase bound ATP, ADP, or Pi from the catalytic site, indicating that binding of ATP to the second and third sites failed to promote release of products from the first site. Direct monitoring of nucleotide binding in the alphaR376A and alphaR376K mutant F(1) by a tryptophan in place of betaTyr-331 (Weber et al. (1993) J. Biol. Chem. 268, 20126-20133) showed that the catalytic sites of the mutant enzymes, like the wild type, have different affinities and therefore, are structurally asymmetric. These results indicate that alphaArg-376, which is close to the beta- or gamma-phosphate group of bound ADP or ATP, respectively, does not make a significant contribution to the catalytic reaction, but coordination of the arginine to nucleotide filling the low-affinity sites is essential for promotion of rotational catalysis to steady-state turnover.
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PMID:Escherichia coli ATP synthase alpha subunit Arg-376: the catalytic site arginine does not participate in the hydrolysis/synthesis reaction but is required for promotion to the steady state. 1070 30

The electron paramagnetic resonance (EPR), electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectra of Mg2+-depleted chloroplast F1-ATPase substituted with stoichiometric VO2+ are reported. The ESEEM and HYSCORE spectra of the complex are dominated by the hyperfine and quadrupole interactions between the VO2+ paramagnet and two different nitrogen ligands with isotropic hyperfine couplings /A1/ = 4.11 MHz and /A2/ = 6.46 MHz and nuclear quadrupole couplings e2qQ1 approximately 3.89-4.49 MHz and e2qQ2 approximately 1.91-2.20 MHz, respectively. Aminoacid functional groups compatible with these magnetic couplings include a histidine imidazole, the epsilon-NH2 of a lysine residue, and the guanidinium group of an arginine. Consistent with this interpretation, very characteristic correlations are detected in the HYSCORE spectra between the 14N deltaM1 = 2 transitions in the negative quadrant, and also between some of the deltaM1 = 1 transitions in the positive quadrant. The interaction of the substrate and product ADP and ATP nucleotides with the enzyme has been studied in protein complexes where Mg2+ is substituted for Mn2+. Stoichiometric complexes of Mn x ADP and Mn x ATP with the whole enzyme show distinct and specific hyperfine couplings with the 31P atoms of the bonding phosphates in the HYSCORE (ADP, A(31Pbeta) = 5.20 MHz: ATP, A(31Pbeta) = 4.60 MHz and A(31Pgamma) = 5.90 MHz) demonstrating the role of the enzyme active site in positioning the di- or triphosphate chain of the nucleotide for efficient catalysis. When the complexes are formed with the isolated alpha or beta subunits of the enzyme, the HYSCORE spectra are substantially modified, suggesting that in these cases the nucleotide binding site is only partially structured.
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PMID:The role of the Mg2+ cation in ATPsynthase studied by electron paramagnetic resonance using VO2+ and Mn2+ paramagnetic probes. 1072 46

Bovine IF(1), a basic protein of 84 amino acids, is involved in the regulation of the catalytic activity of the F(1) domain of ATP synthase. At pH 6.5, but not at basic pH values, it inhibits the ATP hydrolase activity of the enzyme. The oligomeric state of bovine IF(1) has been investigated at various pH values by sedimentation equilibrium analytical ultracentrifugation and by covalent cross-linking. Both techniques confirm that the protein forms a tetramer at pH 8, and below pH 6.5, the protein is predominantly dimeric. By covalent cross-linking, it has been found that at pH 8.0 the fragment of IF(1) consisting of residues 44-84 forms a dimer, whereas the fragment from residues 32-84 is tetrameric. Therefore, some or all of the residues between positions 32 and 43 are necessary for tetramer formation and are involved in the pH-sensitive interconversion between dimer and tetramer. One important residue in the interconversion is histidine 49. Mutation of this residue to lysine abolishes the pH-dependent activation-inactivation, and the mutant protein is active and dimeric at all pH values investigated. It is likely from NMR studies that the inhibitor protein dimerizes by forming an antiparallel alpha-helical coiled-coil over its C-terminal region and that at high pH values, where the protein is tetrameric, the inhibitory regions are masked. The mutation of histidine 49 to lysine is predicted to abolish coiled-coil formation over residues 32-43 preventing interaction between two dimers, forcing the equilibrium toward the dimeric state, thereby freeing the N-terminal inhibitory regions and allowing them to interact with F(1).
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PMID:Modulation of the oligomerization state of the bovine F1-ATPase inhibitor protein, IF1, by pH. 1083 97

Rotation of the F(0)F(1) ATP synthase gamma subunit drives each of the three catalytic sites through their reaction pathways. The enzyme completes three cycles and synthesizes or hydrolyzes three ATP for each 360 degrees rotation of the gamma subunit. Mutagenesis studies have yielded considerable information on the roles of interactions between the rotor gamma subunit and the catalytic beta subunits. Amino acid substitutions, such as replacement of the conserved gammaMet-23 by Lys, cause altered interactions between gamma and beta subunits that have dramatic effects on the transition state of the steady state ATP synthesis and hydrolysis reactions. The mutations also perturb transmission of specific conformational information between subunits which is important for efficient conversion of energy between rotation and catalysis, and render the coupling between catalysis and transport inefficient. Amino acid replacements in the transport domain also affect the steady state catalytic transition state indicating that rotation is involved in coupling to transport.
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PMID:Molecular mechanisms of rotational catalysis in the F(0)F(1) ATP synthase. 1083 45

Atypical protein kinase C-iota (aPKCiota) plays an important role in mitogenic signaling, actin cytoskeleton organization, and cell survival. Apart from the differences in the regulatory domain, the catalytic domain of aPKCiota differs considerably from other known kinases, because it contains a modification within the glycine-rich loop motif (GXGXXG) that is found in the nucleotide-binding fold of virtually all nucleotide-binding proteins including PKCs, Ras, adenylate kinase, and the mitochondrial F1-ATPase. We have used site-directed mutagenesis and kinetic analysis to investigate whether these sequence differences affect the nucleotide binding properties and catalytic activity of aPKCiota. When lysine 274, a residue essential for ATP binding and activity conserved in most protein kinases, was replaced by arginine (K274R mutant), aPKCiota retained its normal kinase activity. This is in sharp contrast to results published for any other PKC or even distantly related kinases like phosphoinositide 3-kinase gamma, where the same mutation completely abrogated the kinase activity. Furthermore, the sensitivity of aPKCiota for inhibition by GF109203X, a substance acting on the ATP-binding site, was not altered in the K274R mutant. In contrast, replacement of Lys-274 by tryptophan (K274W) completely abolished the kinase activity of PKCiota. In accordance with results obtained with other kinase-defective PKC mutants, in cultured cells aPKCiota-K274W acted in a dominant negative fashion on signal transduction pathways involving endogenous aPKCiota, whereas the effect of the catalytically active K274R mutant was identical to the wild type enzyme. In summary, aPKCiota differs from classical and novel PKCs also in the catalytic domain. This information could be of significant value for the development of specific inhibitors of aPKCiota as a key factor in central signaling pathways.
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PMID:Unique structural and functional properties of the ATP-binding domain of atypical protein kinase C-iota. 1090 26

The antibiotic bicyclomycin inhibits rho-dependent termination processes by interfering with RNA translocation by preventing RNA binding at the translocation site or by uncoupling the translocation process from ATP hydrolysis. Previous studies have shown that bicyclomycin binds near the ATP hydrolysis pocket on rho. The hexameric structure of rho indicates that it is in a class of enzymes with strong sequence similarity to F(1)-ATP synthase. The bicyclomycin derivative 5a-formylbicyclomycin, an inhibitor comparable to bicyclomycin, was previously shown to form a stable imine with rho and when reduced to the amine with NaBH(4) to singly label five of the six rho subunits. Lysine-336 was identified by mass spectrometric analysis of trypsin-digested fragments as the site of 5a-formylbicyclomycin adduction. A model of rho was made by threading the rho sequence on the known crystal structure of the alpha and beta subunits of F(1)-ATP synthase. The model, along with information concerning the extent and site of 5a-formylbicyclomycin adduction, indicates an overall C6 symmetry for rho subunit organization. We propose that the sequence similarity between rho and F(1)-ATP synthase extends to a similar quaternary structure and an equivalent enzyme mechanism. The proposed mechanism of RNA translocation coupled with ATP hydrolysis changes the overall symmetry of rho from C6 to C6/C3.
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PMID:Rho transcription factor: symmetry and binding of bicyclomycin. 1092

The correlation between protein molecular weight and the number of lysine or basic amino acid residues was found to be high for broad range molecular weight standards, subunits of Escherichia coli F1F0-ATP synthase and the translated open reading frame of E. coli. A relatively poor correlation between protein molecular weight and the number of cysteine residues was observed in all cases. The ability of amine-reactive, thiol-reactive and basic amino acid-binding fluorophores to detect the eight subunits of F1F0-ATP synthase complex was assessed using 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), monobromobimane (MBB) and SYPRO Ruby protein gel stain, respectively. Though experimentally none of the fluorophores provided accurate estimates of the subunit stoichiometry of this complex, MDPF and SYPRO Ruby protein gel stain were capable of semiquantitative detection of every subunit. MBB, however, failed to detect subunits a, b and c of the hydrophobic F0 complex, as well as subunit epsilon of the F1 complex. All three fluorescent detection procedures permitted subsequent identification of representative subunits by peptide mass profiling using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The use of thiol-reactive fluorophores for the global analysis of protein expression profiles does not appear to be advisable as a significant number of proteins have few or no cysteine residues, thus escaping detection.
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PMID:Comparison of three different fluorescent visualization strategies for detecting Escherichia coli ATP synthase subunits after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 1168 Aug 98

The interface between the c-subunit oligomer and the a subunit in the F0 sector of the ATP synthase is believed to form the core of the rotating motor powered by the protonic flow. Besides the essential cAsp61 and aArg210 residues (Escherichia coli numbering), a few other residues at this interface, although nonessential, show a high degree of conservation, among these aGlu219. The homologous residue aGlu210 in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus has been substituted by a lysine. Inner membranes prepared from the mutant strain showed approximately half of the ATP synthesis activity when driven both by light and by acid-base transitions. As estimated with the ACMA assay, proton pumping rates in the inner membranes were also reduced to a similar extent in the mutant. The most striking impairment of ATP synthesis in the mutant, a decrease as low as 12 times as compared to the wild-type, was observed in the absence of a transmembrane electrical membrane potential (Delta(phi)) at low transmembrane pH difference (Delta(pH)). Therefore, the mutation seems to affect both the mechanism responsible for coupling F1 with proton translocation by F0, and the mechanism determining the relative contribution of Delta(pH) and Delta(phi) in driving ATP synthesis.
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PMID:A point mutation in the ATP synthase of Rhodobacter capsulatus results in differential contributions of Delta(pH) and Delta(phi) in driving the ATP synthesis reaction. 1195 1


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