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 alpha 3 beta 3 hexamer was reconstituted from the alpha and beta subunits of TF1 portion of ATP synthase of thermophilic bacterium (Kagawa et al. (1989) FEBS Lett. 249, 67). The alpha 1 beta 1 heterodimer of ATP synthase was isolated by high performance liquid chromatography (HPLC) of the alpha 3 beta 3 hexamer in the presence of AT(D)P-Mg. On polyacrylamide gel electrophoresis, both bands corresponding to the dimer and hexamer showed ATPase activity. The alpha 1 beta 1 dimer was dissociated into the equal amounts of the alpha and beta monomers by sodium dodecyl sulfate. The alpha and beta monomers were practically inactive. The alpha 2 and beta 2 homodimers were not detected by electrophoresis and HPLC.
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PMID:The alpha 1 beta 1 heterodimer of ATP synthase. 214 34

Functional compatibility between the F1 and F0 parts of ATP synthases from Escherichia coli (EF1F0) and the thermophilic bacterium PS3 (TF1F0) was analyzed. F1-stripped everted membrane vesicles from both organisms bound the homologous or heterologous F1 part to the same extent. Titration of the reconstituted membrane vesicles with dicyclohexylcarbodiimide revealed a similar sensitivity of the homologous and hybrid F1F0 complexes towards the inhibitor. Furthermore, the heterologous enzymes exhibited ATP-dependent H+ translocation comparable to that of homologous F1F0. Antisera raised against EF1 or subunits a, b, and c of EF0 were analyzed for cross-reactivity with TF1 and TF0. Common antigenic sites have been detected with immunoblot analysis for subunit beta and subunit c of EF1F0 and the corresponding subunits from TF1F0. A weak binding of the anti-a and anti-b antisera with the TF0 part has been observed in an enzyme-linked immunosorbent assay. Based on these findings the structural and functional relationship between the mesophilic and thermophilic ATP synthase complexes is discussed.
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PMID:Structural and functional relationship of ATP synthases (F1F0) from Escherichia coli and the thermophilic bacterium PS3. 243 18

The ATPase from the ATP synthase of the thermophilic bacterium PS3 (TF1), unlike F1 ATPase from other sources, does not retain bound ATP, ADP, and Pi at a catalytic site under conditions for single-site catalysis [Yohda, M., & Yoshida, M. (1987) J. Biochem. 102, 875-883]. This raised a question as to whether catalysis by TF1 involved alternating participation of catalytic sites. The possibility remained, however, that there might be transient but catalytically significant retention of bound reactants at catalytic sites when the medium ATP concentration was relatively low. To test for this, the extent of water oxygen incorporation into Pi formed by ATP hydrolysis was measured at various ATP concentrations. During ATP hydrolysis at both 45 and 60 degrees C, the extent of water oxygen incorporation into the Pi formed increased markedly as the ATP concentration was lowered to the micromolar range, with greater modulation observed at 60 degrees C. Most of the product Pi formed arose by a single catalytic pathway, but measurable amounts of Pi were formed by a pathway with high oxygen exchange. This may result from the presence of some poorly active enzyme. The results are consistent with sequential participation of three catalytic sites on the TF1 as predicted by the binding change mechanism.
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PMID:F1 ATPase from the thermophilic bacterium PS3 (TF1) shows ATP modulation of oxygen exchange. 253 Oct 4

Previously we reported that ATPase activity was recovered when the subunit alpha + beta + gamma or alpha + beta + delta of the F1-ATPase from the thermophilic bacterium PS3 were combined under appropriate conditions. Unlike that of holoenzyme (TF1) and the alpha + beta + gamma mixture, ATPase activity of the alpha + beta + delta mixture was heat labile and insensitive to azide inhibition (Yoshida, M., Sone, N., Hirata, H., and Kagawa, Y. (1977) J. Biol. Chem. 252, 3480-3485). Here, the properties of purified subunit complexes were compared in detail with those of native TF1. The subunit stoichiometries of the complexes were determined to be alpha 3 beta 3 gamma 1 and alpha 3 beta 3 delta 1. In general, the properties of the alpha 3 beta 3 gamma complex are very similar to those of TF1, whereas those of the alpha 3 beta 3 delta complex are significantly different. ATPase activity of the alpha 3 beta 3 delta complex is cold labile. The alpha 3 beta 3 delta complex showed a less stringent specificity for substrate and divalent cation than TF1 and the alpha 3 beta 3 gamma complex. Two Km values for ATP were exhibited by the alpha 3 beta 3 delta complex with the lower one being in the range of 0.1 microM. Equilibrium dialysis experiments revealed that the alpha 3 beta 3 delta complex cannot specifically bind ADP in the absence of Mg2+, while TF1 and the alpha 3 beta 3 gamma complex bind about 1 and 3 mol of ADP/mol of enzyme, respectively. ADP-dependent inactivation of the alpha 3 beta 3 delta complex by dicyclohexylcarbodiimide was not observed. The alpha 3 beta 3 gamma complex was readily formed when the gamma subunit was added to the alpha 3 beta 3 delta complex, suggesting that the alpha 3 beta 3 delta complex is not a "dead-end" complex. The cause of thermolability of the alpha 3 beta 3 delta complex appears to be the low stability of the complex itself at high temperature and not due to an unusually low thermostability of the delta subunit.
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PMID:The reconstituted alpha 3 beta 3 delta complex of the thermostable F1-ATPase. 253 13

The highly dissociable alpha 3 beta 3 subunit complex (Mr = 319,582) of thermophilic ATP synthase was crystallized on a mercury surface under oxygen. The two-dimensional crystal was compared with that of TF1 (Mr = 385,351, alpha 3 beta 3 gamma delta epsilon subunit complex) by means of computer image processing. The crystals showed the same hexagonal lattice (a = b = 10 nm), despite the difference in their molecular weights. The color images of the two protein molecules were also hexagonal. However, there was an open hole in the image of the alpha 3 beta 3 complex, where small subunits (gamma, delta, and epsilon) of TF1 may have been located. The structure of this heterohexamer is consistent with that deduced from other physical parameters.
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PMID:Hexagonal structure of two-dimensional crystals of the alpha 3 beta 3 complex thermophilic ATP synthase. 269 17

F1-ATPase is the major enzyme for ATP synthesis in mitochondria, chloroplasts, and bacterial plasma membranes. F1-ATPase obtained from thermophilic bacterium PS3 (TF1) is the only ATPase which can be reconstituted from its primary structure. Its beta subunit constitutes the catalytic site, and is capable of forming hybrid F1's with E. coli alpha and gamma subunits. Since the stability of TF1 resides in its primary structure, we cloned a gene coding for TF1, and the primary structure of the beta subunit was deduced from the nucleotide sequence of the gene to compare the sequence with those of beta's of three major categories of F1's; prokaryotic membranes, chloroplasts, and mitochondria. The following results were obtained. Homology: The primary structure of the TF1 beta subunit (473 residues, Mr = 51,995.6) showed 89.3% homology with 270 residues which are identical in the beta subunits from human mitochondria, spinach chloroplasts, and E. coli. It contained regions homologous to several nucleotide-binding proteins. Secondary structure: The deduced alpha-helical (30.1%) and beta-sheet (22.3%) contents were consistent with those determined from the circular dichroism spectra. Residues forming reverse turns (Gly and Pro) were highly conserved among the F1 beta subunits. Substituted residues and stability of TF1: We compared the amino acid sequence of the TF1 beta subunit with those of the other F1 beta subunits mentioned above. The observed substitutions in the thermophilic subunit increased its propensities to form secondary structures, and its external polarity to form tertiary structure. Codon usage: The codon usage of the TF1 beta gene was found to be unique. The changes in codons that achieved these amino acid substitutions were much larger than those caused by minimal mutations, and the third letters of the optimal codons were either guanine or cytosine, except in codons for Gln, Lys, and Glu.
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PMID:Stable structure of thermophilic proton ATPase beta subunit. 288 Aug 41

Single-site catalysis by F1-ATPase from a thermophilic bacterium PS3 (TF1) was examined by incubating the enzyme with a submolar amount of radioactive ATP. The profile of single-site catalysis by TF1 at 23 degrees C was different from that of beef heart mitochondrial F1-ATPase (MF1). ATP hydrolysis on the enzyme and release of the products was rapid, and subsequent addition of non-radioactive ATP (cold chase) did not promote the hydrolysis of radioactive ATP, indicating that the rate-limiting step was not the step of product release but the step of ATP binding to the enzyme. Thus, the characteristic features of so-called uni-site catalysis were not observed. At 60 degrees C, whether in the presence or absence of phosphate ion, a small amount of bound [alpha, gamma-32P]ATP and cold chase promotion were observed. However, since bound 32P1 was not detected by centrifugal gel filtration, it is not yet certain whether TF1 has typical uni-site characteristics. Based on the hydrolytic turnover rate for single-site catalysis and analysis of the kinetics of steady-state catalysis, it is proposed that single-site catalysis is dominant even in steady-state catalysis at ATP concentrations of less than about 20 microM.
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PMID:Single-site catalysis of F1-ATPase from thermophilic bacterium PS3 and its dominance in steady-state catalysis at low ATP concentration. 289 90

Purified TF1 (F1-ATPase from a thermophilic bacterium PS3) synthesizes enzyme-bound ATP from medium Pi and enzyme-bound ADP in the presence of 50% dimethylsulfoxide (DMSO). Once ATP was formed on the enzyme, it was not released even after removal of DMSO and Pi from the solution. The half maximal concentration of medium Pi for ATP synthesis was 1mM. The pH optimum for enzyme-bound ATP formation was about 6.5. Under the optimum conditions, a yield of up to 0.8 mol of ATP/mol of TF1 was obtained.
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PMID:The synthesis of enzyme-bound ATP by the F1-ATPase from the thermophilic bacterium PS3 in 50% dimethylsulfoxide. 622 32

The soluble F1-ATPase from the thermophilic bacterium PS3 (TF1) contains no endogenous adenine nucleotides and contains about 0.2 g ions of Mg2+/mol which resists removal by repeated centrifugation-elution on columns of Sephadex G-50. The isolated enzyme will not bind additional Mg2+ added in the absence of adenine nucleotides nor is the rate of inactivation of the isolated enzyme by dicyclohexylcarbodiimide (DCCD) affected by the addition of Mg2+. When ADP is added to isolated TF1, a 1:1 TF1 X ADP complex is formed which is stable to repeated gel permeation on columns of Sephadex G-50 subjected to centrifugation-elution. On formation of the 1:1 TF1 X ADP complex, the rate of inactivation of the enzyme by DCCD is accelerated 6-fold. The rate of inactivation of the 1:1 TF1 X ADP complex by DCCD is not further stimulated in the presence of 2 mM ADP which indicates that the binding of ADP to a single site in the enzyme is sufficient to promote maximal stimulation of the inactivation. Addition of Mg2+ to the 1:1 TF1 X ADP complex results in the binding of about 1 g ion of Mg2+/mol of enzyme. The 1:1:1 TF1 X ADP X Mg2+ complex thus formed is sluggishly inactivated by DCCD. When the Mg2+ is removed from the TF1 X ADP X Mg2+ complex by treatment with trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, the rate of inactivation of the enzyme by DCCD is accelerated 4-fold. Other divalent metal ions protect the 1:1 TF1 X ADP complex against inactivation by DCCD. Of these, Mn2+, Zn2+, Co2+, and Cd2+, which are about as equally effective as Mg2+ as cofactors for the hydrolytic reaction when present at 0.2 mM, offer about equal protection of the complex against inactivation by DCCD also when present at 0.2 mM. These results indicate that the binding site for ADP in the 1:1 TF1 X ADP complex is a catalytic site. TF1, inactivated by 92% with DCCD, has the same capacity to bind ADP as the active enzyme, forming a tight 1:1 TF1 X ADP complex which is stable to repeated centrifugation-elution on columns of Sephadex G-50. The 1:1 TF1 X ADP complex retains its capacity to bind Mg2+ to form the 1:1:1 TF1 X ADP X Mg2+ complex after it is inactivated by 88% with DCCD.
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PMID:Modulation by ADP and Mg2+ of the inactivation of the F1-ATPase from the thermophilic bacterium, PS3, with dicyclohexylcarbodiimide. 622 24

Eadie-Hofstee plots (v/[S] vs. v) of the kinetics of ATP hydrolysis by purified bovine heart mitochondrial F1-ATPase (MF1) over a substrate (MgATP) concentration range of 1-5000 microM were curvilinear, indicating negative cooperativity with respect to [MgATP] as originally shown by Ebel & Lardy (1975) [Ebel, R. E., & Lardy, H. A. (1975) J. Biol. Chem. 250, 191-196]. The data were computer analyzed for the best fit of the least number of straight lines, each representing a different apparent Km and Vmax. The best fits for MF1 and TF1 from the thermophilic bacterium PS3 were three lines in each case. The upper limits of the apparent Km values for MF1 were of the order of 10(-6), 10(-4), and 10(-3) M, and the corresponding apparent Vmax values (per minute per milligram of protein) were in the range of micromoles or less for the lowest Km line and decamicromoles for the other two. The results for TF1 were very similar. The presence of an activating anion (10 mM KHCO3) in the MF1 assay medium increased the overall Vmax by about 50% and eliminated the high Km but had essentially no effect on the intermediate and low Km's, indicating retention of negative cooperativity in the corresponding substrate concentration range. Kinetic data for MgITP as substrate also yielded two Km values (in the absence of KHCO3) differing by about 10(4)-fold. The relationship between [14C]dicyclohexylcarbodiimide [( 14C]-DCCD) binding to MF1 and activity inhibition was linear up to approximately 1 mol of DCCD bound/mol of MF1. At this point, the degree of inhibition was about 95%.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetics of ATP hydrolysis by F1-ATPase and the effects of anion activation, removal of tightly bound nucleotides, and partial inhibition of the ATPase by covalent modification. 623 25


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