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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Seventeen mutations in beta-subunit of Escherichia coli F1-ATPase which had previously been characterized in strain AN1272 (Mu-induced mutant) were expressed in strain JP17 (beta-subunit gene deletion). Six showed unchanged behavior, namely: C137Y; G142D; G146S; G207D; Y297F; and Y354F. Five failed to assemble F1F0 correctly, namely: G149I; G154I; G149I,G154I; G223D; and P403S,G415D. Six assembled F1F0 correctly, but with membrane ATPase lower than in AN1272, namely: K155Q; K155E; E181Q; E192Q; D242N; and D242V. AN1272 was shown to unexpectedly produce a small amount of wild-type beta-subunit; F1-ATPase activities reported previously in AN1272 were referable to hybrid enzymes containing both mutant and wild-type beta-subunits. Purified F1 was obtained from K155Q; K155E; E181Q; E192Q; and D242N mutants in JP17. Vmax ATPase values were lower, and unisite catalysis rate and equilibrium constants were perturbed to greater extent, than in AN1272. However, general patterns of perturbation revealed by difference energy diagrams were similar to those seen previously, and the new data correlated well in linear free energy relationships for reaction steps of unisite catalysis. Correlation between multisite and unisite ATPase activity was seen in the new enzymes. Overall, the data give strong support to previously proposed mechanisms of unisite catalysis, steady-state catalysis, and energy coupling in F1-ATPases (Al-Shawi, M. K., Parsonage, D. and Senior, A. E. (1990) J. Biol. Chem. 265, 4402-4410). The K155Q, K155E, D242N, and E181Q mutations caused 5000-fold, 4000-fold, 1800-fold, and 700-fold decrease, respectively, in Vmax ATPase, implying possibly direct roles for these residues in catalysis. Experiments with the D242N mutant suggested a role for residue beta D242 in catalytic site Mg2+ binding.
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PMID:Further examination of seventeen mutations in Escherichia coli F1-ATPase beta-subunit. 140 Apr 60

Fluorescent 5-coordinate organotin-flavone complexes of 3-hydroxy-flavone (Hof) and 3,5,7,2',4',-pentahydroxyflavone (morin) are good inhibitors of mitochondrial F1F0ATPase but do not inhibit F1-ATPase and they have been examined as possible fluorescent probes of F1F0ATPase. R2SnX (morin) complexes exhibit low fluorescence enhancement on binding to mitochondrial membranes with no displacement by equimolar tributyltin. In contrast R2SnX (of) complexes exhibit high fluorescence enhancement whose extent is variable and is displacable by equimolar tributyltin. Fluorescence enhancement by R2SnX (of) complexes correlates with the ATPase I50 values. Dialkyltin-3-hydroxy flavone, R2SnX(of), complexes act as a new class of fluorescent probes which titrate the F0 segment of F1F0ATPase.
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PMID:Organotin-flavone complexes: a new class of fluorescent probes for F1F0ATPase. 141 56

The presence of ATP at non-catalytic sites of the chloroplast F1-ATPase (CF1) eliminates a considerable lag in onset of enzyme activity that otherwise occurs in the presence of bicarbonate [Milgrom, Y. M., Ehler, L. & Boyer, P. D. (1991) J. Biol. Chem. 266, 11551-11558]. Sulfite is known to be much more effective than bicarbonate in stimulating ATPase activity CF1. Results reported here show that when assayed in the presence of sulfite, CF1, with some non-catalytic sites empty or filled with GT(D)P, is able to hydrolyze both ATP and GTP. Thus, the presence of adenine nucleotides at non-catalytic sites is not necessary for catalytic turnover of CF1. However, even though CF1 with empty non-catalytic sites shows a significant initial activity, the prior binding of adenine nucleotides at non-catalytic site(s) results in further activation of MgATPase and MgGTPase activities, even at relatively high sulfite and substrate concentrations. Although extensive activation of CF1 results from the presence of sulfite, with or without nucleotide binding at non-catalytic sites, the Km remains constant, at about 50 microM for MgATP and 400 microM for MgGTP. The results obtained show that the ATPase activity of CF1 is determined by the fraction of the active enzyme. The inactive CF1.ADP.Mg2+ formed during MgATP hydrolysis can be rapidly trapped by azide to provide a measure of the fraction of inactive enzyme. Increasing the concentration of sulfite increases the fraction of active CF1 in the assay medium. Measurements with radioactively labeled nucleotides show that the presence of ATP at non-catalytic sites promotes the ATP-dependent release of inhibitory ADP from a catalytic site. The activating effect of ATP binding at non-catalytic sites results from increasing the portion of CF1 in an active state during steady-state ATP hydrolysis.
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PMID:The mechanism of stimulation of MgATPase activity of chloroplast F1-ATPase by non-catalytic adenine-nucleotide binding. Acceleration of the ATP-dependent release of inhibitory ADP from a catalytic site. 142 75

The half-life of the F1-ATPase beta-subunit (F1-beta) mRNA in ATPase-poor brown adipose tissue (BAT) (t1/2 = 9.5 h) was found to be 3-7-fold shorter than in liver (t1/2 = 27 h) and heart (t1/2 = 63 h) of mice. When translated in reticulocyte lysate, a 2-3-fold lower efficiency appeared with F1-beta mRNA from BAT than from other tissues. The in vitro synthesized F1-beta protein precursors of BAT, liver and heart origin were imported and processed by mouse liver mitochondria with equal efficiency. The results indicate that the pool of abundant F1-beta mRNA in BAT is not fully translatable, most likely due to its low metabolic stability.
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PMID:Low translational efficiency of the F1-ATPase beta-subunit mRNA largely accounts for the decreased ATPase content in brown adipose tissue mitochondria. 142 64

F1 (alpha beta) complexes containing equimolar ratios of the alpha and beta subunits have been shown to function as active ATPases, whereas individually isolated alpha and beta subunits show no real ATPase activity. These results indicate that the single-copy subunits are not required for F1-ATPase activity. The minimal F1 (alpha beta)-core complexes exhibit, however, lower rates and some different properties from those of their parent whole F1 or alpha 3 beta 3 gamma complexes. It is therefore concluded that for obtaining a full spectrum of the characteristic functional properties of an F1-ATPase the presence of the F1-gamma subunit is also required. The implications of these findings on the subunit location of both catalytic and noncatalytic nucleotide binding sites is discussed.
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PMID:Identification of subunits required for the catalytic activity of the F1-ATPase. 142 38

A 490 bp DNA fragment was amplified from Methanosarcina barkeri genomic DNA by the polymerase chain reaction (PCR) using oligonucleotide primers designed based on conserved amino acid sequences of the F1-ATPase beta subunits. The amino acid sequence deduced from the DNA sequence of this fragment was highly homologous to a portion of the F1-ATPase beta subunit. This indicates that this archaebacterium has a gene of F-type ATPase in addition to a gene of V-type ATPase.
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PMID:A DNA fragment homologous to F1-ATPase beta subunit was amplified from genomic DNA of Methanosarcina barkeri. Indication of an archaebacterial F-type ATPase. 146 48

(1) Dimethyl sulfoxide (DMSO) markedly inhibited the Vmax of multisite ATPase activity in Escherichia coli F1-ATPase at concentrations greater than 30% (v/v). Vmax/KM was reduced by 2 orders of magnitude in 40% (v/v) DMSO at pH 7.5, primarily due to reduction of Vmax. The inhibition was rapidly reversed on dilution into aqueous buffer. (2) KdATP at the first, high-affinity catalytic site was increased 1500-fold from 2.3 x 10(-10) to 3.4 x 10(-7) M in 40% DMSO at pH 7.5, whereas KdADP was increased 3.2-fold from 8.8 to 28 microM. This suggests that the high-affinity catalytic site presents a hydrophobic environment for ATP binding in native enzyme, that there is a significant difference between the conformation for ADP binding as opposed to ATP binding, and that the ADP-binding conformation is more hydrophilic. (3) Rate constants for hydrolysis and resynthesis of bound ATP in unisite catalysis were slowed approximately 10-fold by 40% DMSO; however, the equilibrium between bound Pi/bound ATP was little changed. The reduction in catalysis rates may well be related to the large increase in KdATP (less constrained site). (4) Significant Pi binding to E. coli F1 could not be detected either in 40% DMSO or in aqueous buffer using a centrifuge column procedure. (5) We infer, on the basis of the measured constants KaATP, K2 (hydrolysis/resynthesis of ATP), k+3 (Pi release), and KdADP and from estimates of k-3 (Pi binding) that delta G for ATP hydrolysis in 40% DMSO-containing pH 7.5 buffer is between -9.2 and -16.8 kJ/mol.
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PMID:Effects of dimethyl sulfoxide on catalysis in Escherichia coli F1-ATPase. 153 Oct 28

A soluble form of the b subunit of the F0 sector of the F1F0-ATPase of Escherichia coli has been produced, purified, and characterized. In this form of the protein, designated bsol, residues 25-146 (the carboxyl terminus) of b have been fused to an amino-terminal octapeptide extension derived from the vector pUC8. The inferred subunit molecular weight of bsol is 15,459. bsol protein was expressed in E. coli as a soluble cytoplasmic protein and was readily purified to homogeneity by conventional methods. The molecular weight of bsol, determined by sedimentation equilibrium, was 31,200, indicating that the protein is dimeric. Chemical cross-linking studies supported this conclusion. However, bsol sedimented with a coefficient of just 1.8 S and behaved on size exclusion chromatography with an apparent molecular weight of 80,000-85,000. These results indicate that the protein exists in solution as a highly elongated dimer. The circular dichroism spectrum indicated that bsol is highly alpha-helical. Binding of bsol to F1-ATPase was directly demonstrated by size exclusion chromatography. bsol also inhibited the binding of F1-ATPase to F1-depleted membrane vesicles, as measured by reconstitution of energy-dependent quinacrine fluorescence quenching. This result implies that bsol and F0 compete for binding to the same site on F1. The apparently normal interaction of bsol with F1-ATPase strongly suggests that the recombinant protein assumes the correct structure. No substantial effects of bsol on the ATPase activity of purified F1 were observed.
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PMID:The polar domain of the b subunit of Escherichia coli F1F0-ATPase forms an elongated dimer that interacts with the F1 sector. 153 97

(1) Previous mutational analyses have shown that residue beta R398 of the beta-subunit is a key residue for binding of the inhibitory antibiotic aurovertin to Escherichia coli F1Fo-ATP synthase. Here, we studied purified F1 from the beta R398C and beta R398W mutants. ATPase activity in both cases was resistant to aurovertin inhibition. The fluorescence spectrum (lambda exc = 278 or 295 nm) of beta R398W F1 showed a significant red-shift as compared to wild-type and beta R398C enzymes, indicating that residue beta R398 lies in a polar environment. On the basis of this and previous evidence, we propose that aurovertin binding to F1-ATPase involves a specific charged donor-acceptor H-bond between residue beta R398 and the 7-hydroxyl group of aurovertin. (2) The fluorescent substrate analog lin-benzo-ADP was shown to bind to beta R398W F1 catalytic sites with the same Kd values as to wild-type F1, and with the same quenching of the fluorescence of the analog. Fluorescence energy transfer was seen between the beta R398W residue and bound lin-benzo-ADP. Analysis of transfer efficiency at varying stoichiometry of bound lin-benzo-ADP showed that interaction occurred between one beta R398W residue and one catalytic-site-bound analog molecule at a distance of approximately 23 A. The relationships of the aurovertin and catalytic sites in the primary and tertiary structure are discussed.
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PMID:Investigation of the aurovertin binding site of Escherichia coli F1-ATPase by fluorescence spectroscopy and site-directed mutagenesis. 153 96

Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.
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PMID:Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha. 167 60


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