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 amino acid sequence -Gly-X-X-X-X-Gly-Lys- occurs in many, diverse, nucleotide-binding proteins, and there is evidence that it forms a flexible loop which interacts with one or other of the phosphate groups of bound nucleotide. This sequence occurs as -Gly-Gly-Ala-Gly-Val-Gly-Lys- in the beta-subunit of the enzyme F1-ATPase, where it is thought to form part of the catalytic nucleotide-binding domain. Mutants of Escherichia coli were generated in which residue beta-lysine 155, at the end of the above sequence, was replaced by glutamine or glutamate. Properties of the soluble purified F1-ATPase from each mutant were studied. The results showed: 1) replacement of lysine 155 by Gln or Glu decreased the steady-state rate of ATP hydrolysis by 80 and 66%, respectively. 2) Characteristics of ATP hydrolysis at a single site were not markedly changed in the mutant enzymes, implying that lysine 155 is not directly involved in bond cleavage during ATP hydrolysis or bond formation during ATP synthesis. 3) The binding affinity for MgATP was weakened considerably in the mutants (Lys much much greater than Gln greater than Glu), whereas the binding affinity for MgADP was affected only mildly (Lys = Gln greater than Glu), suggesting that lysine 155 interacts with the gamma-phosphate of ATP bound at a single high affinity catalytic site. 4) The major determinant of inhibition of steady-state ATPase turnover rate in the mutant enzymes was an attenuation of positive catalytic cooperativity. 5) The data are consistent with the idea that during multisite catalysis residue 155 of beta-subunit undergoes conformational movement which changes substrate and product binding affinities.
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PMID:Directed mutations of the strongly conserved lysine 155 in the catalytic nucleotide-binding domain of beta-subunit of F1-ATPase from Escherichia coli. 289 6

Pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), an adenine nucleotide affinity analog, was found to bind in a saturable fashion to isolated alpha-subunit from Escherichia coli F1-ATPase with a stoichiometry of one mol/mol and a Kd approximately 150 microM. The binding was shown to be specific by the following criteria: 1) ATP reduced the binding of PLP-AMP by 80%, and 2) PLP-AMP, like ATP, induced a conformational change which increased the mobility of alpha-subunit in nondenaturing polyacrylamide gel electrophoresis and rendered alpha-subunit resistant to mild trypsin proteolysis. A stable adduct was formed between isolated alpha-subunit and [3H] PLP-AMP after reduction with NaBH4. alpha-Subunit labeled to the extent of 0.4-0.7 mol/mol was digested with trypsin and subjected to high pressure liquid chromatography purification, yielding a single labeled peptide. Automated amino acid sequencing showed that residue alpha-Lys-201 was specifically labeled. The results suggest that Lys-201 occupies a position proximate to the phosphate groups of bound ATP in the alpha.ATP complex. PLP-AMP did not support repolymerization of isolated alpha-, beta-, and gamma-subunits, consistent with previous reports that subunit repolymerization in vitro is dependent upon the presence of nucleoside triphosphate. Further, PLP-AMP-labeled alpha-subunit could not be reconstituted with isolated beta- and gamma-subunits in the presence of ATP, showing that occupation of the alpha-subunit nucleotide site by PLP-AMP impairs normal subunit-subunit interaction.
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PMID:Pyridoxal 5'-diphospho-5'-adenosine binds at a single site on isolated alpha-subunit from Escherichia coli F1-ATPase and specifically reacts with lysine 201. 289 72

The rates of hydrolysis of acetyl phosphate in the presence of 0.1 M NaOH and of ATP in the presence of either 1 M HCl or 1 M NaOH were measured at different temperatures and in the presence of different concentrations of the organic solvents dimethyl sulfoxide or ethylene glycol. Under all conditions tested, there was a progressive increase in the rate constant of hydrolysis of both phosphate compounds as the water activity of the medium was decreased by the addition of organic solvents. At 25 degrees C, substitution of 70% of the water of the medium by dimethyl sulfoxide promoted an increase of two orders of magnitude in the rate constant of acetyl phosphate hydrolysis. In the presence of 80% and 90% dimethyl sulfoxide the rate of acetyl phosphate hydrolysis increased by more than two orders of magnitude and was so fast that it could not be measured with the method used. The effect of organic solvents on the rate of ATP hydrolysis was less pronounced than that observed for acetyl phosphate hydrolysis. At 30 degrees C, substitution of 90% of water by an organic solvent promoted a 4-6-fold increase of the rate of ATP hydrolysis. Acceleration of either acetyl phosphate or ATP hydrolysis rates was promoted by a decrease in both activation energies (Ea) and in entropies of activation delta S. The data obtained are discussed with reference to the mechanism of catalysis of enzymes involved in energy transduction such as the Ca2+-ATPase of sarcoplasmic reticulum and the F1-ATPase of mitochondria.
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PMID:Role of water activity on the rates of acetyl phosphate and ATP hydrolysis. 289 8

Binding of about 1 mol of adenosine triphosphopyridoxal to Escherichia coli F1-ATPase resulted in the nearly complete inactivation of the enzyme [(1987) J. Biol. Chem. 262, 7686-7692]. About two thirds of the label was bound to the alpha-subunit, and the rest to the beta-subunit. The present study revealed that Lys201 in the alpha-subunit and Lys155 in the glycine-rich region of the beta-subunit are the major sites labeled with this reagent. Thus, these two residues might be located close to the gamma-phosphate of the bound ATP.
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PMID:Identification of alpha-subunit Lys201 and beta-subunit Lys155 at the ATP-binding sites in Escherichia coli F1-ATPase. 289 87

Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F1 ATPase (CF1) is bound at catalytic sites or at noncatalytic regulatory sites or both has been uncertain. We have used photolabeling by 2-azido-ATP and 2-azido-ADP to ascertain the location, with Mg2+ activation, of tightly bound ADP (a) that inhibits the hydrolysis of ATP by chloroplast ATP synthase, (b) that can result in an inhibited form of CF1 that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF1. The data show that in all instances the inhibition is associated with ADP binding without inorganic phosphate (Pi) at catalytic sites. After photophosphorylation of ADP or 2-azido-ADP with [32P]Pi, similar amounts of the corresponding triphosphates are present on washed thylakoid membranes. Trials with appropriately labeled substrates show that a small portion of the tightly bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling by an ADP moiety at a catalytic site. We also report the occurrence of a 1-2-min delay in the onset of the Mg2+-induced inhibition after addition of CF1 to solutions containing Mg2+ and ATP, and that this delay is not associated with the filling of noncatalytic sites. A rapid burst of Pi formation is followed by a much lower, constant steady-state rate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Relationship of tightly bound ADP and ATP to control and catalysis by chloroplast ATP synthase. 290 55

The alpha-subunit of Escherichia coli F1-ATPase contains an adenine-specific noncatalytic nucleotide-binding domain. A recent proposal (Maggio, M. B., Pagan, J., Parsonage, D., Hatch, L., and Senior, A. E. (1987) J. Biol. Chem. 262, 8981-8984) suggested that this domain is formed by residues 160-340, approximately, in alpha-subunit. Within this proposed domain is a sequence Gly-X-X-X-X-Gly-Lys which is conserved in a large and diverse group of nucleotide-binding proteins and is thought to interact with phosphate groups of bound nucleotide. In this work, residue alpha Lys-175, the terminal residue of the above conserved sequence in F1-alpha-subunit, was mutagenized to Ile or Glu. The specific activity of purified mutant F1-ATPase was reduced by 2.5-fold (Ile) or 3-fold (Glu). Apparent binding of ATP to alpha-subunit, as measured by the centrifuge column procedure, was strongly impaired and ATP-induced conformational change in alpha-subunit, as measured by protection against trypsin proteolysis, was nearly abolished in both mutants. The results suggest that residue alpha Lys-175 is located within the nucleotide-binding domain of alpha-subunit, and that this residue is functionally involved in nucleotide binding. The results support previous suggestions that the alpha-subunit nucleotide-binding site is not involved, directly or indirectly, in catalysis.
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PMID:Directed mutagenesis of the strongly conserved lysine 175 in the proposed nucleotide-binding domain of alpha-subunit from Escherichia coli F1-ATPase. 290 46

Carbon and phosphorus metabolism of cell suspensions of Methanosarcina barkeri strain MS (DSM 800), grown on methanol, were probed in vivo by NMR. The experimental conditions, which involved thick cell suspensions, did not significantly affect the efficiency of the rate of methanol uptake by cells. Following exposure to methanol an acidification of both the intracellular and the extracellular spaces was observed and a gradient of 0.5 pH units across the cytoplasmic membrane was determined from the 31P-NMR data. High levels of intracellular ATP up to 4 mM were detected. The ADP concentration determined in a suspension of starved cells was only 2 mM, suggesting that a significant amount of ADP may be immobilized and is thus not detectable by NMR. In the presence of the protonophore, 3,3',4',5-tetrachlorosalicylanilide, the proton gradient was dissipated and the synthesis of ATP stopped. The inhibitor of the ATP synthase, N,N'-dicyclohexylcarbodiimide, was rather inefficient in inhibiting ATP synthesis. High concentrations of N,N'-dicyclohexylcarbodiimide (corresponding to 300 nmol/mg protein-1) were required to decrease the ATP content by approximately 60%, and, under these conditions, formation of acetyl phosphate was detected. However, the methanol consumption rate was not affected.
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PMID:In vivo 31P- and 13C-NMR studies of ATP synthesis and methane formation by Methanosarcina barkeri. 292 75

Psychosine, sphingosylphosphorylcholine (52-104 microM), and other glycosphingolipids stimulate mitochondrial respiration (up to 500%) and inhibit oxidative phosphorylation to varying degrees. Above 104 microM these functions as well as uptake of Ca2+ are prevented. At 104 microM sphingosylphosphorylcholine inhibits the mitochondrial ATPase reaction in submitochondrial particles by 48%. Both sphingosylphosphorylcholine and psychosine enhance the active phosphate-dependent swelling of mitochondria. Passive swelling occurs in the presence of rotenone (when swelling does not normally occur) and under hypotonic conditions. A direct interaction of sphingosylphosphorylcholine with membranes is demonstrated by a discharge of the proton gradient across mitochondrial membranes, hemolysis of red blood cells, and binding to inner and outer mitochondrial membranes. Thus lysosphingolipids bind strongly to mitochondrial membranes and markedly alter mitochondrial function. This alteration would affect the ATP levels, thereby altering a wide range of ATP-dependent cellular functions. These results offer a partial explanation for the pathogenesis of representative lysosomal storage diseases.
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PMID:Lysosphingolipids and mitochondrial function. II. Deleterious effects of sphingosylphosphorylcholine. 297 68

We have used 31P nuclear magnetic resonance (NMR) techniques to characterize bioenergetic changes in the Langendorff-perfused rat heart accompanying alterations in thyroid state. Cytosolic phosphocreatine and inorganic phosphate concentrations changed significantly in both the hypo- and hyperthyroid groups compared to controls; the calculated phosphorylation potential [( ATP]/[ADP][Pi]) increased by 60% in hypothyroidism and decreased by 60% in hyperthyroidism relative to the euthyroid value of 47 X 10(3) M-1. Creatine phosphokinase (CPK) and mitochondrial ATP synthase rates were measured in the intact tissue using a saturation-transfer NMR method. There were no significant differences in the measured fluxes through the CPK reaction among the three groups (4.24 +/- 1.00 mM X sec-1 for the euthyroid group). Although O2 consumption increased by 46% in hearts from hyperthyroid animals, no change in the measured mitochondrial ATP synthase flux was observed compared to the euthyroid flux of 1.05 +/- 0.11 mM X sec-1. These results suggest that the apparent in situ P/O ratio of mitochondria in hearts from hyperthyroid animals is reduced relative to that in euthyroid controls.
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PMID:A phosphorus-31 nuclear magnetic resonance study of effects of altered thyroid state on cardiac bioenergetics. 298 61

The effects of fenoctimine, an inhibitor of gastric acid secretion, on the microsomal (H+ + K+)-ATPase were studied. In the micromolar concentration range, fenoctimine inhibited hydrolysis of ATP and p-nitrophenyl phosphate by the (H+ + K+)-ATPase. Inhibition was reversible and noncompetitive with substrate. The apparent Ki was dependent on the concentration of membranes, being increased by added liposomes or high microsomal membrane concentrations. Over the concentration range that (H+ + K+)-ATPase was inhibited, fenoctimine increased the turbidity of microsomal suspensions. The effects of fenoctimine were not specific for the gastric (H+ + K+)-ATPase, since the hydrolytic activities of the (Na+ + K+)-ATPase and mitochondrial ATPase were also inhibited by the drug. These results suggest that inhibition of hydrolysis may not be the direct result of an interaction between the (H+ + K+)-ATPase and fenoctimine but the secondary effect of a fenoctimine-induced perturbation of the microsomal membrane.
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PMID:Inhibition of the gastric (H+ + K+)-ATPase by fenoctimine. 299 Apr 81


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