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)

ATP hydrolysis by the Escherichia coli F1 ATPase (ECF1) induces a conformational change in the gamma subunit. This change can be monitored by fluorescence changes in N-[4-[7-(diethylamino)-4-methyl]coumarin-3-yl)]maleimide (CM) bound at a cysteine introduced by site-directed mutagenesis into the gamma subunit at position 106 [Turina, P., & Capaldi, R. A. (1994) J. Biol. Chem. 269, 13465-13471]. In studies reported here, the magnitude of the fluorescence change has been determined with the noncleavable nucleotide analogue AMP-PNP and by rapid measurements using the slowly cleavable ATP gamma S. The data indicate that maximal fluorescence change occurs with binding of 1 mol of nucleotide triphosphate per mole of ECF1. During unisite catalysis, ATP binding causes a fluorescence enhancement from CM bound at position 106, which is then followed by fluorescence quenching. The kinetics of these fluorescence changes have been measured using both ATP and ATP gamma S as substrate. With ATP gamma S, these kinetics can be simulated using rate constants similar to those for ATP except for an approximately 30-fold slower rate of the bond cleavage and resynthesis steps, i.e., k+2 and k-2. The observed rates and amplitudes of the fluorescence changes on hydrolysis of ATP and ATP gamma S were analyzed by simulations in which the bond cleavage or the Pi release step was responsible for fluorescence quenching. The results indicate that ATP or ATP gamma S binding causes the fluorescence enhancement of CM bound to the gamma subunit and that this conformational change is reversed upon bond cleavage to yield ADP.Pi or ADP.PiS in catalytic sites.
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PMID:ATP binding causes a conformational change in the gamma subunit of the Escherichia coli F1ATPase which is reversed on bond cleavage. 794 38

Second messenger regulation of IRK1 (Kir2.1) inward rectifier K+ channels was investigated in giant inside-out patches from Xenopus oocytes. Kir2.1-mediated currents that run down completely within minutes upon excision of the patches could be partly restored by application of Mg-ATP together with > 10 microM free Mg2+ to the cytoplasmic side of the patch. As restoration could not be induced by the ATP analogs AMP-PNP or ATP gamma S, this suggests an ATPase-like mechanism. In addition to ATP, the catalytic subunit of cAMP-dependent protein kinase (PKA) induced an increase in current amplitude, which could, however, only be observed if channels were previously or subsequently stimulated by Mg-ATP and free Mg2+. This indicates that functional activity of Kir2.1 channels requires both phosphorylation by PKA and ATP hydrolysis. Moreover, currents could be down-regulated by N-heptyl-5-chloro-1-naphthalenesulfonamide, a specific stimulator of protein kinase C (PKC), suggesting that PKA and PKC mediate inverse effects on Kir2.1 channels. Regulation of Kir2.1 channels described here may be an important mechanism for regulation of excitability.
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PMID:Kir2.1 inward rectifier K+ channels are regulated independently by protein kinases and ATP hydrolysis. 799 32

The uncoupling of Ca2+ transport from ATP hydrolysis in the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by trypsin digestion was re-investigated by comparing ATPase activity with the ability of the enzyme to occlude Eu3+ (a transport parameter) after various tryptic digests. With this method, re-examination of uncoupling by tryptic digest of the ATPase revealed that TD2 cleavage (Arg-198) had no effect on either occlusion or ATPase activity. Digestion past TD2 in the presence of 5 mM Ca2+ and at 25 degrees C resulted in the loss of about 70% of the ATPase activity, but no loss of occlusion. Digestion past TD2 in the presence of 5 mM Ca2+, 3 mM ATP, and at 25 degrees C resulted in a partially uncoupled enzyme complex which retained about 50% of the ATPase activity, but completely lost the ability to occlude Eu3+. Digest past TD2 in the presence of 5 mM Ca2+ and 3 mM AMP-PNP (a non-hydrolyzable ATP analog) at 25 degrees C resulted in no loss of occlusion, thus revealing the absolute requirement of ATP during the digest to eliminate occlusion. From these findings we conclude that uncoupling of Ca2+ transport from ATPase activity is possible by tryptic digestion of the (Ca2+ + Mg2+)-ATPase. Interestingly, only after phosphorylation of the enzyme do the susceptible bond(s) which lead to the loss of occlusion become exposed to trypsin.
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PMID:Uncoupling of occlusion from ATP hydrolysis activity in sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase. 800 38

Nucleotide-induced structural rearrangements of MalK, the ATP-hydrolyzing component of the ATP binding cassette transporter for maltose from Salmonella typhimurium were investigated by means of analysis of intrinsic tryptophan fluorescence and limited proteolysis. ATP was found to decrease the tryptophan fluorescence of purified MalK by 37 +/- 1%. ADP or adenosine 5'-O-(3-(thio)triphosphate) (ATP gamma S) caused similar quenching while AMP was rather ineffective. Mg2+ ions were not required. Exposure of MalK to increasing concentrations of trypsin and subsequent analysis by SDS-polyacrylamide gel electrophoresis and immunoblotting revealed the formation of three major transiently stable peptide fragments of 24 (T2), 23 (T3), and 20 kDa (T4), respectively. In addition, a minor rapidly degraded fragment of 33 kDa (T1) was observed. However, in the presence of MgATP, fragment T1 as well as a substantial fraction of native MalK were strongly protected against proteolytic attack. Similar protection against the protease was observed in the presence of MgGTP or, to a lesser extent, MgCTP. In contrast, MgADP, ATP in the presence of EDTA, CaATP or nonhydrolyzable nucleotides such as MgATP gamma S or MgAMP-PNP (beta, gamma-imidoadenosine-5'-triphosphate) failed to significantly affect the susceptibility of MalK to the protease. MgATP similarly affected the tryptic digestion pattern of a mutant protein (MalKK42R) that exhibits only a much reduced ATPase activity but has retained the capability to bind nucleotides. N-terminal protein sequence analysis of the peptides revealed cleavage by trypsin at Arg66 (T1), Arg146 (T2), Arg153 (T3), and Arg185 (T4), respectively. These results indicate that (i) nucleotide binding to MalK is accompanied by a global conformational change of the protein; (ii) a very specific interaction occurs with substrates of the MalK-ATPase, resulting in structural changes that involve the helical domain from Arg66 to Arg146; and (iii) the C-terminal half of MalK is rather resistant to proteolysis.
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PMID:Nucleotide-induced conformational changes of MalK, a bacterial ATP binding cassette transporter protein. 805 Nov 43

The interactions of the 70-kDa heat-shock proteins (hsp70s) with their protein substrates appear to be regulated by bound nucleotide. Previous work has shown that the nucleotide binding site of the bovine brain uncoating ATPase, a constitutive member of the hsp70 family, crystallographically resembles the nucleotide binding site of actin and, like actin, the uncoating ATPase has a strongly bound ADP which cannot be removed by dialysis or treatment with ethylenediaminetetraacetic acid (EDTA). This suggests that, like the bound nucleotide of actin, it may be required for the enzyme to retain its native structure. In this study, the strongly bound ADP was removed by first replacing it with 5'-adenylyl imidodiphosphate (AMP-PNP) and then removing the bound AMP-PNP by dialysis. Following this treatment, more than 95% of the uncoating ATPase becomes nucleotide-free. The nucleotide-free uncoating ATPase retains its ability to bind and hydrolyze ATP and to uncoat clathrin-coated vesicles, even after 10 days of storage at 4 degrees C. Therefore, in contrast to actin, the bound nucleotide of the uncoating ATPase is not required to prevent denaturation of the enzyme. Using nucleotide-free uncoating ATPase, we were able to accurately measure the dissociation constants of ATP, ADP, and the nucleotide analogues AMP-PNP and 2'-deoxyadenosine 5'-triphosphate (dATP). The dissociation constants of both ATP and ADP are about 10(-8) M, more than 1-2 orders of magnitude stronger than previously reported, while AMP-PNP and dATP bind 2-3 orders of magnitude more weakly than ATP.
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PMID:Characterization of nucleotide-free uncoating ATPase and its binding to ATP, ADP, and ATP analogues. 811 62

Transcription of vaccinia virus early genes in vitro requires the virally encoded RNA polymerase and early transcription factor, VETF. VETF is a promoter-binding protein with DNA-dependent ATPase activity. We have investigated the functional role of VETF in transcription activation by analyzing the interaction between the RNA polymerase and promoter DNA. Using a gel shift assay, a novel protein-DNA complex was detected that required both RNA polymerase and VETF. The complex was suggested to be a transcription initiation complex by its ability to incorporate 32P-labeled nucleotides in combinations compatible with synthesis of a short RNA chain. Competition binding studies indicated that the RNA polymerase associated specifically with a viral early promoter. These experiments demonstrate that VETF activates transcription by directly recruiting the RNA polymerase to the promoter. Sedimentation analysis showed that VETF and RNA polymerase did not form a stable complex unless promoter DNA was present, indicating that protein-protein contacts are not the sole basis for initiation complex assembly. DNase I cleavage and methylation interference analyses revealed a hyperreactive site in the center of the promoter. Radiolabeling of RNA in the RNA polymerase-promoter complex did not occur when AMP-PNP (adenyl-5'-yl imidodiphosphate) was substituted for ATP, suggesting that ATP hydrolysis is required for the initiation of transcription. A model is proposed to account for these findings.
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PMID:Recruitment of vaccinia virus RNA polymerase to an early gene promoter by the viral early transcription factor. 842 51

Tentoxin, a cyclic tetrapeptide produced by the fungus Alternaria tenuis, is a potent inhibitor of the chloroplast coupling factor 1 from certain sensitive species of plants. We have shown that the beta subunit is at least partly responsible for conferring sensitivity to the toxin. This was confirmed by Avni et al. (Avni, A., Anderson, J.D., Holland, N., Rochaix, J-D., Gromet-Elhanan, Z., and Edelman, M. (1992) Science 257, 1245-1247) who demonstrated the importance for tentoxin sensitivity of an acidic amino acid residue at position 83 in the beta subunit sequence. In this paper we show that the Ca(2+)-ATPase and Mg(2+)-ATPase activities of CF1 lacking the delta and epsilon subunits, CF1(-delta epsilon), were fully sensitive to tentoxin, even after the gamma subunit is cleaved by trypsin into several smaller fragments. We also show that the isolated reconstitutively active beta subunit of CF1 does not effectively compete with CF1(-delta epsilon) for tentoxin binding. The results suggest that tight tentoxin binding requires the presence of at least the alpha and beta subunits but is independent of the delta and epsilon subunits. Tentoxin inhibited the release of a tightly bound molecule of ADP from CF1, which was induced by the binding of the ATP analogue adenylyl-beta,gamma-imidodiphosphate (AMP-PNP). AMP-PNP was shown previously (Shapiro, A.B., and McCarty, R.E. (1990) J. Biol. Chem. 265, 4340-4347) to cause two adenine nucleotide binding sites on CF1, sites 1 and 3, to switch their properties, possibly as part of an alternating site catalytic cooperativity mechanism (Boyer, P.D. (1989) FASEB J. 3, 2164-2178). It is proposed that the effect of tentoxin on catalytic cooperativity in CF1 results from tentoxin binding at an interface between alpha and beta subunits, preventing transfer of information between different nucleotide binding sites on the enzyme.
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PMID:Inhibition by tentoxin of cooperativity among nucleotide binding sites on chloroplast coupling factor 1. 847 98

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

Under resting conditions, steady-state [Ca] in agonist-sensitive Ca stores reflects a balance between active uptake (usually mediated by a thapsigargin-sensitive Ca-ATPase of the SERCA family) and passive efflux of Ca. Even though this pump-leak cycle appears to be a common property of Ca-storing organelles, little is known about the nature of the leak pathway. Ca homeostasis in thapsigargin-sensitive internal Ca stores of single permeabilized BHK-21 fibroblasts was examined using digital image processing of compartmentalized mag-fura-2 (a low-affinity Ca indicator). It is shown here that the leak of Ca from internal stores is regulated specifically by the cytosolic ATP concentration. The rate of leak was 3.6 times slower in 0.375 mM[ATP] than in 4 mM [ATP] (Na or Mg salt). These effects were observed in the presence of 0 Ca/EGTA, thapsigargin, heparin, and ruthenium red, and therefore appear to be independent of the Ca-ATPase, the InsP(3) receptor and the ryanodine receptor. The ATP-stimulated leak was seen in a variety of cell types, including rat basophilic leukemia cells and mouse pancreatic acinar cells. Other nucleotides (ADP, GTP, CTP, and UTP) and nonhydrolyzable ATP analogs (AMP-PNP and ATPgammaS) did not reproduce the action of ATP. Changes in cellular metabolism and ensuing alterations in [ATP] will be expected to influence the filling state of internal Ca stores through effects on the passive leak pathway, potentially leading to modulation of Ca signaling and organellar function.
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PMID:ATP regulates calcium leak from agonist-sensitive internal calcium stores. 864 63

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


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