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)

We introduced mutations to test the function of the conserved amino-terminal region of the gamma subunit from the Escherichia coli ATP synthase (F0F1-ATPase). Plasmid-borne mutant genes were expressed in an uncG strain which is deficient for the gamma subunit (gamma Gln-14-->end). Most of the changes, which were between gamma Ile-19 and gamma Lys-33, gamma Asp-83 and gamma Cys-87, or at gamma Asp-165, had little effect on growth by oxidative phosphorylation, membrane ATPase activity, or H+ pumping. Notable exceptions were gamma Met-23-->Arg or Lys mutations. Strains carrying these mutations grew only very slowly by oxidative phosphorylation. Membranes prepared from the strains had substantial levels of ATPase activity, 100% compared with wild type for gamma Arg-23 and 65% for gamma Lys-23, but formed only 32 and 17%, respectively, of the electrochemical gradient of protons. In contrast, other mutant enzymes with similar ATPase activities (including gamma Met-23-->Asp or Glu) formed H+ gradients like the wild type. Membranes from the gamma Arg-23 and gamma Lys-23 mutants were not passively leaky to protons and had functional F0 sectors. These results suggested that substitution by positively charged side chains at position 23 perturbed the energy coupling. The catalytic sites of the mutant enzymes were still regulated by the electrochemical H+ gradient but were inefficiently coupled to H+ translocation in both ATP-dependent H+ pumping and delta mu H+ driven ATP synthesis.
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PMID:F0F1-ATPase gamma subunit mutations perturb the coupling between catalysis and transport. 140 Mar 98

Arg-210 of the a subunit of the Escherichia coli F0F1-ATPase has been proposed previously as a component of the proton pore. A mutant in which lysine was substituted for Arg-210 was generated and was found to be unable to translocate protons. A plasmid carrying this mutation, along with wild-type genes encoding the c and b subunits, was unusual in that it failed to complement a chromosomal c-subunit mutation on succinate minimal medium. Three revertants on succinate minimal medium contained plasmids that showed complementation with chromosomal c-subunit but not with a-subunit mutations. One of these had a deletion in the a subunit. The other two were point mutations, resulting in the substitution of aspartic acid by Gly-53 and of arginine for Leu-211. The Gly-53 to aspartic acid change implied that Gly-53 and Arg-210 are normally in close proximity. To test this idea further, a series of mutants in which aspartic acid was placed in helix I at positions ranging from 42 to 57 was generated. Full complementation was regained only when the aspartic acid residue was present on the same side of a putative helix as Gly-53 over a span of three turns of the alpha-helix. These results and others suggest modifications of a previously proposed model for the transmembrane helices of the F0 portion of the F0F1-ATPase. The implications of these modifications for the mechanism of proton translocation are discussed.
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PMID:Second-site revertants of an arginine-210 to lysine mutation in the a subunit of the F0F1-ATPase from Escherichia coli: implications for structure. 140 2

Use of the nonphosphorylating beta,gamma-bidentate chromium(III) complex of ATP to induce a stable Ca(2+)-occluded form of the sarcoplasmic reticulum Ca(2+)-ATPase was combined with molecular sieve high performance liquid chromatography of detergent-solubilized protein to examine the ability of the Ca(2+)-ATPase mutants Gly-233-->Glu, Gly-233-->Val, Glu-309-->Gln, Gly-310-->Pro, Pro-312-->Ala, Ile-315-->Arg, Leu-319-->Arg, Asp-703-->Ala, Gly-770-->Ala, Glu-771-->Gln, Asp-800-->Asn, and Gly-801-->Val to occlude Ca2+. This provided a new approach to identification of amino acid residues involved in Ca2+ binding and in the closure of the gates to the Ca2+ binding pocket of the Ca(2+)-ATPase. The "phosphorylation-negative" mutant Asp-703-->Ala and mutants of ADP-sensitive phosphoenzyme intermediate type were fully capable of occluding Ca2+, as was the mutant Gly-770-->Ala. Mutants in which carboxylic acid-containing residues in the putative transmembrane segments had been substituted ("Ca(2+)-site mutants") and mutant Gly-801-->Val were unable to occlude either of the two calcium ions. In addition, the mutant Gly-310-->Pro, previously classified as ADP-insensitive phosphoenzyme intermediate type (Andersen, J.P., Vilsen, B., and MacLennan, D.H. (1992). J. Biol. Chem. 267, 2767-2774), was unable to occlude Ca2+, even though Ca(2+)-activated phosphorylation from MgATP took place in this mutant.
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PMID:CrATP-induced Ca2+ occlusion in mutants of the Ca(2+)-ATPase of sarcoplasmic reticulum. 146 90

The potassium-translocating Kdp-ATPase of Escherichia coli shares common functional properties with eukaryotic P-type ATPases. The KdpB subunit has been identified as the catalytic subunit forming the phosphorylated intermediate. Substitution of Asp-307 in KdpB by Glu, Asn, Gln, Tyr, His, Ala or Ser by site-directed mutagenesis and the subsequent transfer of the point mutations to the chromosome revealed that the mutants were not functioning with respect to cell growth at low K+ concentrations and ATPase activity as well as phosphorylation capacity of the purified Kdp complex. These findings indicate that Asp-307 in KdpB is the phosphorylation site of the Kdp-ATPase. In contrast, replacement of the close but non-conserved Asp-300 by Asn or Glu has no immediate influence on the enzyme functions tested. However, the Km for K+ of the ATPase activity has been increased 30-fold compared with the wild-type enzyme.
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PMID:The phosphorylation site of the Kdp-ATPase of Escherichia coli: site-directed mutagenesis of the aspartic acid residues 300 and 307 of the KdpB subunit. 147 95

The roles of the Escherichia coli H(+)-ATPase (FoFl) delta subunit (177 amino acid residues) was studied by analyzing mutants. The membranes of nonsense (Gln-23----end, Gln-29----end, Gln-74----end) and missense (Gly-150----Asp) mutants had very low ATPase activities, indicating that the delta subunit is essential for the binding of the Fl portion to Fo. The Gln-176----end mutant had essentially the same membrane-bound activity as the wild type, whereas in the Val-174----end mutant most of the ATPase activity was in the cytoplasm. Thus Val-174 (and possibly Leu-175 also) was essential for maintaining the structure of the subunit, whereas the two carboxyl terminal residues Gln-176 and Ser-177 were dispensable. Substitutions were introduced at various residues (Thr-11, Glu-26, Asp-30, Glu-42, Glu-82, Arg-85, Asp-144, Arg-154, Asp-161, Ser-163), including apparently conserved hydrophilic ones. The resulting mutants had essentially the same phenotypes as the wild type, indicating that these residues do not have any significant functional role(s). Analysis of mutations (Gly-150----Asp, Pro, or Ala) indicated that Gly-150 itself was not essential, but that the mutations might affect the structure of the subunit. These results suggest that the overall structure of the delta subunit is necessary, but that individual residues may not have strict functional roles.
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PMID:Escherichia coli H(+)-ATPase: role of the delta subunit in binding Fl to the Fo sector. 153 Sep 99

Illumination of sarcoplasmic reticulum vesicles by ultraviolet light in the presence of 1 mM vanadate causes photocleavage of the Ca(2+)-ATPase into two fragments (Vegh et al. (1990) Biochim. Biophys. Acta 1023, 168-183). In the absence of Ca2+ the photocleavage occurs in the N-terminal half of the molecule near the phosphate acceptor Asp-351. In the presence of 2 mM Ca2+ the photocleavage shifts to the C-terminal half of the ATPase, near the FITC binding site (Lys-515). About half of the Ca(2+)-ATPase was cleaved rapidly, accompanied by nearly complete, irreversible loss of ATPase activity when illuminated in the presence of 2 mM CaCl2; further cleavage of the enzyme was slow and affected primarily the C-terminal fragment produced in the presence of Ca2+. Solubilization of the Ca(2+)-ATPase with C12E8 did not affect the site of photocleavage in either conformation. The vanadate-induced Ca(2+)-ATPase crystals were disrupted during photocleavage, while the binding of anti-ATPase antibodies directed against the phosphorylation site (PR-8) and against the FITC binding region (PR-11) was enhanced. The bovine kidney Na+,K(+)-ATPase was insensitive to photocleavage under conditions where about half the Ca(2+)-ATPase was fragmented. The slight cleavage of the pig gastric H+,K(+)-ATPase after prolonged illumination produced fragments that are distinct from the fragments of the Ca(2+)-ATPase.
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PMID:Differences in the susceptibility of various cation transport ATPases to vanadate-catalyzed photocleavage. 165 3

N-(1-Pyrene)maleimide is a hydrophobic, sulfhydryl-directed, chemical modification probe which, at a low concentration, inhibits the capacity of lamb kidney sodium- and potassium-activated adenosine triphosphatase [Na,K)-ATPase; EC 3.6.1.3) to bind ouabain. This inhibition is partially blocked by preincubation of the enzyme with ouabagenin, an aglycone derivative which can be used as a reversible protecting ligand for the ouabain binding site. The kinetics of inhibition are not first order, suggesting that there may be more than one site of labeling which is responsible for the inhibition of ouabain binding. Although earlier work (Kirley, T. L., Lane, L. K., and Wallick, E. T. (1986) J. Biol. Chem. 261, 4525-4528) indicates that the inhibition is accompanied by a loss in the number of binding sites rather than a decrease in affinity of the sites for the ligand, other data (Scheiner-Bobis, G., Zimmerman, M., Kirch, V., and Schoner, W. (1987) Eur. J. Biochem. 165, 653-656) indicates that there is no cysteine residue located extracellularly in the ouabain binding site. By sequence analysis of alpha subunit peptides labeled by N-(1-pyrene)maleimide in the absence but not in the presence of protecting ligand, it is demonstrated in this work that there are two major sites of labeling protected by the binding of ouabagenin, Cys-367 and Cys-656. Both of these sites are located in the large cytoplasmic domain of the alpha subunit, one close to the phosphorylation site (Asp-369), and the other implicated in the binding of ATP (Cys-656). Therefore, it appears from this data that the inhibition of ouabain binding by N-(1-pyrene)maleimide is not due to modification of a site in the binding pocket for cardiac glycosides, but rather to an allosteric effect, since cardiac glycoside binding is known to be dependent on the phosphorylation state of the enzyme. The dependence of inhibition on the presence of sodium, potassium, and ATP also is consistent with this interpretation. The work reported here thus explains the apparent paradox posed by the earlier data.
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PMID:Identification of cysteine residues in lamb kidney (Na,K)-ATPase essential for ouabain binding. 165 8

The monoclonal antibody 5-B6, directed against the alpha-subunit of pig gastric H+,K(+)-ATPase (Mg(2+)-dependent H(+)-transporting and K(+)-stimulated ATPase), was shown to be a potent inhibitor of the K(+)-ATPase activity, thereby binding to the cytoplasmic side of the alpha-subunit of the enzyme [Van Uem, Peters & De Pont (1990) Biochim. Biophys. Acta 1023, 56-62]. In order to define the epitope for 5-B6 on pig gastric H+,K(+)-ATPase more precisely, the alpha-subunit of the enzyme was subjected to limited proteolysis followed by chemical cleavage. Restricted proteolysis with papain followed by sequence analysis yielded an immunoreactive fragment of 27 kDa beginning at Ser379. This fragment was water-soluble and possessed the fluorescein isothiocyanate-reaction site. Limited tryptic digestion in the presence of K+ gave rise to an immunoreactive 56 kDa fragment beginning at Ile456, thus restricting the location of the epitope from Ile456 to the C-terminal end of the 27 kDa fragment (around residue 620). Further degradation of the 27 kDa fragment by means of formic acid cleavage at Asp-Pro bonds resulted initially in the formation of two non-immunoreactive fragments of 17 kDa and 11 kDa, indicating that the epitope for 5-B6 has to be localized around the chemical cleavage sites Asp507 and/or Asp510. Comparison of the primary structure of the alpha-subunits of gastric H+,K(+)-ATPase and non-immunoreactive rat kidney Na+,K(+)-ATPase shows almost no similarity for the sequence containing these formic acid cleavage sites (Thr504-Leu-Glu-Asp-Pro-Arg-Asp-Pro-Arg512), whereas the adjacent sequences are nearly 100% identical. These findings strongly suggest that the epitope for 5-B6 includes (part of) this sequence.
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PMID:Determination of the epitope for the inhibitory monoclonal antibody 5-B6 on the catalytic subunit of gastric Mg(2+)-dependent H(+)-transporting and K(+)-stimulated ATPase. 172 Jun 15

We have used fluorescence spectroscopy to characterize three covalently bound spectroscopic maleimide derivatives with respect to their location within the tertiary structure of the Ca-ATPase of sarcoplasmic reticulum (SR). These derivatives include (1) 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid, (2) 4-(dimethylamino)azobenzene-4'-maleimide, and (3) fluorescein 5'-maleimide. Biochemical assays demonstrate that modification with any of these three derivatives results in the same functional effects, observed following derivatization of cysteines 344 and 364 by N-ethylmaleimide [Saito-Nakatsuka et al. (1987) J. Biochem. (Tokyo) 101, 365-376]. These residues bracket the ATPase's phosphorylation site (Asp 351) and thus may provide spectroscopic probes of the protein's conformation in this essential region. In agreement with sequencing results, SDS-polyacrylamide gels show that maleimide-modified SR exhibits fluorescence exclusively on the A1 tryptic fragment of the Ca-ATPase. Extensive tryptic digestion followed by centrifugation demonstrates essentially all of the fluorescence was associated with the soluble rather than insoluble (membrane-associated) peptides, confirming the predicted extramembranous location of these residues. Utilizing frequency-domain fluorescence spectroscopy, we were able to recover the transient effects associated with a distribution of donor-acceptor distances. We find from these fluorescence resonance energy transfer measurements that covalently bound maleimide probes are 36 A apart, independent of whether a discrete distance is assumed or a distance distribution model is utilized, in which the conformational variability of the protein is taken into account. While a unimodal distance distribution is adequate to describe the intensity decay associated with maleimide-directed donor-acceptor pairs, a bimodal distribution of distances is necessary to describe the frequency response associated with the energy transfer between maleimide-directed chromophores and other covalently bound probes on the Ca-ATPase, consistent with the large spatial separation observed between maleimides. We recover mean distances of 42 and 77 A between maleimide sites and bound FITC (Lys 515) and mean distances of 28 and 37 A between the maleimide- and the iodoacetamide-directed probes (Cys 670 and 674, whose close proximity approximates a single locus). The measured distances are presented in a model and have permitted us to describe a unique arrangement of these covalently bound probes within both the secondary and tertiary structure of the Ca-ATPase. The resolution inherent in the frequency-domain fluorescence technique to multiple donor-acceptor distances should be generally applicable to a wide range of biological systems in which specific labeling of single unique donor-acceptor sites is not feasible.
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PMID:Frequency-domain fluorescence spectroscopy resolves the location of maleimide-directed spectroscopic probes within the tertiary structure of the Ca-ATPase of sarcoplasmic reticulum. 182 7

Yeast mutants in which genes encoding subunits of the vacuolar H(+)-ATPase were interrupted were assayed for their vacuolar ATPase and proton-uptake activities. The vacuoles from the mutants lacking subunits A (72 kDa), B (57 kDa), or c (proteolipid, 16 kDa) were completely inactive in these reactions. Immunological studies revealed that in the absence of each one of those subunits the catalytic sector was not assembled. Labeling with N,N'-[14C]dicyclohexylcarbodiimide showed the presence of the proteolipid in vacuoles of mutants in which genes encoding subunits of the catalytic sectors were interrupted. No labeling was detected in the mutant in which the gene encoding the proteolipid was interrupted. We conclude that of all the ATPase subunits only the proteolipid is assembled independently and it serves as a template for the assembly of the other subunits. Site-specific mutations were generated in the gene encoding the proteolipid. All of the drastic changes and replacements gave inactive proteins. About half of the single amino acid replacements gave active proteins. Replacing glutamic acid-137 by any of several amino acids, except for aspartic acid, abolished the activity of the enzyme. Other amino acids that may function in proton conductance were changed. It was found that glycine residues may replace amino acids with exchangeable protons.
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PMID:Mutational analysis of yeast vacuolar H(+)-ATPase. 182 30


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