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)

Comparison of profiles of radioactive peptides resolved by HPLC from tryptic digests of the bovine heart F1-ATPase depleted of nucleotides (nd-MF1) which had been photoinactivated with 2-N3-[beta-32P]ADP, on the one hand, and 2-[8-3H]ADP, on the other, shows that the beta phosphate of ADP tethered to tyrosine-beta 345 is slowly hydrolyzed in the presence of Mg2+. When nd-MF1 was photoinactivated with 2-N3-[8-3H]ADP in the absence of Mg2+, hydrolysis of the beta phosphate from ADP tethered to tyrosine-beta 345 was not observed. Subsequent addition of Mg2+ initiated conversion of ADP tethered to tyrosine-beta 345 to tethered AMP suggesting that functional groups at the catalytic site participate in the hydrolytic reaction.
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PMID:ADP tethered to tyrosine-beta 345 at the catalytic site of the bovine heart F1-ATPase is converted to tethered AMP by Mg(2+)-dependent hydrolysis when the enzyme is photoinactivated with 2-N3-ADP. 801 53

Tryptophan fluorescence was investigated as a tool to study the noncatalytic nucleotide-binding sites of Escherichia coli F1-ATPase. Site-directed mutagenesis, affinity labeling, and lin-benzo-ATP binding studies had shown that residues alpha R365 and beta Y354 are located close to the base moiety of bound nucleotide; here, we mutagenized each to tryptophan. The new tryptophans gave a fluorescence signal indicating an environment of high (alpha W365) or intermediate (beta W354) polarity in unoccupied sites. alpha W365 fluorescence was completely quenched by binding of ATP or ADP, providing a direct, specific probe of noncatalytic site nucleotide occupancy. Using this signal, we measured binding parameters for ATP and ADP, showed that nucleotide binding was magnesium-dependent, and showed that GTP and ITP did bind to some extent, but AMP, GDP, and IDP did not. It was possible to follow initial rates of MgATP hydrolysis and noncatalytic site binding under identical conditions; the results indicated that occupancy of noncatalytic sites was not required for catalysis. Fluorescence from beta W354 was quenched completely by lin-benzo-ATP, but only slightly by ATP or ADP. Probably, residue beta 354 is not as closely juxtaposed to the adenine ring of bound ATP and ADP as is residue alpha 365. With either alpha W365 or beta W354 as donor and catalytic site-bound lin-benzo-ADP as acceptor, no fluorescence resonance energy transfer was detected, indicating that the distance between non-catalytic and catalytic sites is > or = 27 A.
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PMID:Tryptophan fluorescence provides a direct probe of nucleotide binding in the noncatalytic sites of Escherichia coli F1-ATPase. 815 56

Two ATP analogs, 2- and 8-azidoadenyl-5'-yl imidodiphosphate, were synthesized, purified and utilized as inhibitors of soluble beef heart mitochondrial F1-ATPase under non-photolytical conditions. In the range of 5 microM to 3 mM ATP, the initial rates of ATP hydrolysis in the presence and absence of the inhibiting ATP analogs can be adequately described by two pairs of Km and Vmax values (3 microM, 8.5 mumol ATP/min per mg; 255 microM, 42.0 mumol ATP/min per mg). With increasing inhibitor concentrations, the apparent Km,2 increases as in competitive inhibition, while Vmax,1 decreases as in non-competitive inhibition. The Ki values derived for both types of inhibition are similar, but strongly different for 2- and 8-azido-AMP-PNP (4 microM and 460 microM, respectively). The decrease of the high-affinity Vmax is compensated by an increase in low-affinity catalysis, resulting in a constant sum of maximal velocities. These data can be described by a model where two sites interact with negative cooperativity in binding of substrate.
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PMID:Beef heart mitochondrial F1-ATPase: inhibition by azidoadenyl-5'-yl imidodiphosphates and cooperative binding of substrate. 839 86

The rat basophilic cell line RBL-1 is known to express high levels of the Ca2+ current activated by store depletion, known as Ca2+ release-activated Ca2+ current (ICRAC), the main Ca2+ influx pathway so far identified in nonexcitable cells. We show here that, as reported in other cell types, metabolic drugs strongly inhibit the Ca2+ influx operated by store depletion in RBL-1 cells also. We have tested the hypothesis that intracellular adenine and/or guanine nucleotide levels act as coupling factors between ICRAC and cell metabolism. Using the whole cell configuration of the patch-clamp technique, we demonstrate that addition of ADP to the intracellular solution significantly reduces ICRAC induced by inositol 1,4,5-trisphosphate. This phenomenon differs from other regulatory pathways of ICRAC, since it is highly temperature-dependent, is observable only in the presence of low intracellular Ca2+ buffering capacity, and requires a cytosolic factor(s) which is rapidly lost during cell dialysis. Moreover, the inhibition is specific for ADP and is partially mimicked by ADPbetaS and AMP, but not by GDP or GTP.
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PMID:Intracellular ADP modulates the Ca2+ release-activated Ca2+ current in a temperature- and Ca2+-dependent Way. 862 86

The enzyme ATP synthase, or F-ATPase, is present in the membranes of bacteria, chloroplasts and mitochondria. Its structure is bipartite, with a proton-conducting, integral membrane portion, F0, and a peripheral portion, F1. Solubilized F1 is composed of five different subunits, (alpha beta)3 gamma delta epsilon, and is active as an ATPase. The function of F-ATPase is to couple proton translocation through F0 with ATP synthesis in F1 (ref.3). Several lines of evidence support the spontaneous formation of ATP on F1 (refs 4,5) and its endergonic release at cooperative and rotating (or at least alternating) sites. The release of ATP at the expense of protonmotive force might involve mechanical energy transduction from F0 into F1 by rotation of the smaller subunits (mainly gamma) within (alpha beta)3, the catalytic hexagon of F1 as suggested by electron microscopy, by X-ray crystal structure analysis and by the use of cleavable crosslinkers. Here we record an intersubunit rotation in real time in the functional enzyme by applying polarized absorption relaxation after photobleaching to immobilized F1 with eosin-labelled gamma. We observe the rotation of gamma relative to immobilized (alpha beta)3 in a timespan of 100 ms, compatible with the rate of ATP hydrolysis by immobilized F1. Its angular range, which is of at least 200 degrees, favours a triple-site mechanism of catalysis, with gamma acting as a crankshaft in (alpha beta)3. The rotation of gamma is blocked when ATP is substituted with its non-hydrolysable analogue AMP-PNP.
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PMID:Intersubunit rotation in active F-ATPase. 863 1

Cys-87, one of two intrinsic cysteines of the gamma subunit of the Escherichia coli ATP synthase (ECF1F0), is in a short segment of this subunit that binds to the bottom domain of a beta subunit close to a glutamate (Glu-381). Cys-87 was unreactive to maleimides under all conditions in wild-type ECF1 and ECF1F0 but became reactive when Glu-381 of beta was replaced by a cysteine or alanine. The reactivity of Cys-87 with maleimides was nucleotide-dependent, occurring with ATP or ADP + EDTA in catalytic sites, in the presence of AMP.PNP + Mg2+ but not with ADP + Mg2+ bound, whether Pi was present or not, and not when nucleotide binding sites were empty. Binding of N-ethylmaleimide had no effect, whereas 7-diethyl-amino-3-(4'-maleimidylphenyl)-4-methylcoumarin increased the ATPase activity of ECF1 more than 2-fold by reaction with Cys-87. In ECF1F0, these reagents inhibited activity. The nucleotide dependence of the reaction of Cys-87 of the gamma subunit depended on the presence of the epsilon subunit. In epsilon subunit-free ECF1, maleimides reacted with Cys-87 under all nucleotide conditions, including when catalytic sites were empty. These results are discussed in terms of nucleotide-dependent movements of the gamma subunit during functioning of the F1F0-type ATPase.
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PMID:Conformational changes in the Escherichia coli ATP synthase (ECF1F0) monitored by nucleotide-dependent differences in the reactivity of Cys-87 of the gamma subunit in the mutant betaGlu-381 --> Ala. 866

In the structure of bovine mitochondrial F1-ATPase that was previously determined with crystals grown in the presence of adenylyl-imidodiphosphate (AMP-PNP) and ADP, the three catalytic beta-subunits have different conformations and nucleotide occupancies. Adenylyl-imidodiphosphate is bound to one beta-subunit (betaTP), ADP is bound to the second (betaDP), and no nucleotide is bound to the third (betaE). Here we show that the uncompetitive inhibitor aurovertin B binds to bovine F1 at two equivalent sites in betaTP and betaE, in a cleft between the nucleotide binding and C-terminal domains. In betaDP, the aurovertin B pocket is incomplete and is inaccessible to the inhibitor. The aurovertin B bound to betaTP interacts with alpha-Glu399 in the adjacent alphaTP subunit, whereas the aurovertin B bound to betaE is too distant from alphaE to make an equivalent interaction. Both sites encompass betaArg-412, which was shown by mutational studies to be involved in binding aurovertin. Except for minor changes around the aurovertin pockets, the structure of bovine F1-ATPase is the same as determined previously. Aurovertin B appears to act by preventing closure of the catalytic interfaces, which is essential for a catalytic mechanism involving cyclic interconversion of catalytic sites.
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PMID:The structure of bovine F1-ATPase complexed with the antibiotic inhibitor aurovertin B. 869 18

In the previously determined structure of mitochondrial F1-ATPase determined with crystals grown in the presence of adenylyl-imidodiphosphate (AMP-PNP) and ADP, the three catalytic beta-subunits have different conformations and nucleotide occupancies. AMP-PNP and ADP are bound to subunits beta TP and beta DP, respectively, and the third beta-subunit (beta E) has no bound nucleotide. The efrapeptins are a closely related family of modified linear peptides containing 15 amino acids that inhibit both ATP synthesis and hydrolysis by binding to the F1 catalytic domain of F1F0-ATP synthase. In crystals of F1-ATPase grown in the presence of both nucleotides and inhibitor, efrapeptin is bound to a unique site in the central cavity of the enzyme. Its binding is associated with small structural changes in side chains of F1-ATPase around the binding pocket. Efrapeptin makes hydrophobic contacts with the alpha-helical structure in the gamma-subunit, which traverses the cavity, and with subunit beta E and the two adjacent alpha-subunits. Two intermolecular hydrogen bonds could also form. Intramolecular hydrogen bonds probably help to stabilize efrapeptin's two domains (residues 1-6 and 9-15, respectively), which are connected by a flexible region (beta Ala-7 and Gly-8). Efrapeptin appears to inhibit F1-ATPase by blocking the conversion of subunit beta E to a nucleotide binding conformation, as would be required by an enzyme mechanism involving cyclic interconversion of catalytic sites.
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PMID:The structure of bovine F1-ATPase complexed with the peptide antibiotic efrapeptin. 879 Mar 45

ATP hydrolyses by the wild-type alpha 3 beta 3 gamma and mutant (alpha D261N)3 beta 3 gamma subcomplexes of the F1-ATPase from the thermophilic Bacillus PS3 have been compared. The wild-type complex hydrolyzes 50 microM ATP in three kinetic phases: a burst decelerates to an intermediate phase, which then gradually accelerates to a final rate. In contrast, the mutant complex hydrolyzes 50 microM or 2 mM ATP in two kinetic phases. The mutation abolishes acceleration from the intermediate phase to a faster final rate. Both the wild-type and mutant complexes hydrolyze ATP with a lag after loading a catalytic site with MgADP. The rate of the MgADP-loaded wild-type complex rapidly accelerates and approaches that observed for the wild-type apo-complex. The MgADP-loaded mutant complex hydrolyzes ATP with a more pronounced lag, and the gradually accelerating rate approaches the slow, final rate observed with the mutant apo-complex. Lauryl dimethylamide oxide (LDAO) stimulates hydrolysis of 2 mM ATP catalyzed by wild-type and mutant complexes 4- and 7.5-fold, respectively. The rate of release of [3H]ADP from the Mg[3H]ADP-loaded mutant complex during hydrolysis of 40 microM ATP is slower than observed with the wild-type complex. LDAO increases the rate of release of [3H]ADP from the preloaded wild-type and mutant complexes during hydrolysis of 40 microM ATP. Again, release is slower with the mutant complex. When the wild-type and mutant complexes are irradiated in the presence of 2-N3-[3H]ADP plus Mg2+ or 2-N3-[3H]ATP plus Mg2+ and azide, the same extent of labeling of noncatalytic sites is observed. Whereas ADP and ATP protect noncatalytic sites of the wild-type and mutant complexes about equally from labeling by 2-N3-[3H]ADP or 2-N3-[3H[ATP, respectively, AMP-PNP provides little protection of noncatalytic sites of the mutant complex. The results suggest that the substitution does not prevent binding of ADP or ATP to noncatalytic sites, but rather that it affects cross-talk between liganded noncatalytic sites and catalytic sites which is necessary to promote dissociation of inhibitory MgADP.
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PMID:The alpha 3 beta 3 gamma complex of the F1-ATPase from thermophilic Bacillus PS3 containing the alpha D261N substitution fails to dissociate inhibitory MgADP from a catalytic site when ATP binds to noncatalytic sites. 884 68

Mild trypsin digestion of isolated bovine-heart mitochondrial F1-ATPase removed the first 15 residues from the N-terminus of subunit alpha under conditions in which other F1 subunits were apparently untouched. When the trypsinized F1 (TF1) was reconstituted with the F0 sector in the mitochondrial membrane (USMP), the ATP hydrolase activity acquired oligomycin sensitivity but ATP hydrolysis was decoupled from proton pumping. TF1 added to USMP did not block the proton channel in F0 as the native F1 did. AMP-PNP inhibited proton conductivity in reconstituted F1-USMP but this effect was lost in reconstituted TF1-USMP. These results indicate that the N-terminus of the F1 alpha subunit plays a critical role in the conformational communication between F1 and F0.
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PMID:The effect of mild trypsin digestion of F1 on energy coupling in the mitochondrial ATP synthase. 895 69


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