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)

Phospholamban is a pentameric transmembrane phosphoprotein that regulates the activity of the Ca(2+)-transporting ATPase (SERCA2a) in cardiac sarcoplasmic reticulum. To better understand the structure and function of phospholamban and its mode of regulation of the ATPase, phospholamban and SERCA2a were coexpressed at high levels in Sf21 insect cells using the baculovirus expression system. SERCA2a was expressed as a functionally active Ca2+ pump, accounting for > or = 20% of the total protein in Sf21 cell microsomes. Wild-type phospholamban, as well as phospholamban with different point mutations in the transmembrane region, inhibited both Ca2+ transport and ATP hydrolysis by the recombinant Ca2+ pump. The inhibition of SERCA2a activity was reversed by an anti-phospholamban monoclonal antibody. The phospholamban molecules studied decreased the apparent Ca2+ affinity of the Ca2+ pump, but had no effect on enzyme velocity measured at saturating Ca2+ concentration. Monomeric phospholamban produced by mutations in the leucine/isoleucine zipper domain decreased the apparent Ca2+ affinity the most, giving stronger inhibition of the Ca2+ pump than even wild-type phospholamban. Thus, the baculovirus cell expression system is ideally suited for examining functional interactions between phospholamban and SERCA2a. The results obtained suggest that the phospholamban monomer may be the active species inhibiting the Ca2+ pump in the cardiac sarcoplasmic reticulum membrane.
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PMID:High-level coexpression of the canine cardiac calcium pump and phospholamban in Sf21 insect cells. 1060 39

In order to test molecular models of cardiac calcium transport regulation, we have used spectroscopy to probe the structures, dynamics, and interactions of the Ca pump (Ca-ATPase) and phospholamban (PLB) in cardiac sarcoplasmic reticulum (SR) and in reconstituted membranes. Electron paramagnetic resonance (EPR) and phosphorescence of probes bound to the Ca pump show that the activity of the pump is quite sensitive to its oligomeric interactions. In cardiac SR, PLB aggregates and inhibits the pump, and both effects are reversed by PLB phosphorylation. Previous analyses of PLB's oligomeric state were only in detergent solutions, so we used EPR and fluorescence to determine the oligomeric structure of PLB in its native state in lipid bilayers. Wild-type PLB is primarily oligomeric in the membrane, while the mutant L37A-PLB is monomeric. For both proteins, phosphorylation shifts the dynamic monomer-oligomer equilibrium toward oligomers, and induces a similar structural change, as indicated by tyrosine fluorescence; yet L37A-PLB is more effective than wild-type PLB in inhibiting and aggregating the pump. Fluorescence energy transfer shows that the Ca pump increases the fraction of monomeric PLB, indicating that the pump preferentially binds monomeric PLB. These results support a reciprocal aggregation model for Ca pump regulation, in which the Ca pump is aggregated and inhibited by association with PLB monomers, and phosphorylation of PLB reverses these effects while decreasing the concentration of PLB monomers. To investigate the structure of the PLB pentamer in more detail, we measured the reactivities of cysteine residues in the transmembrane domain of PLB, and recorded EPR spectra of spin labels attached to these sites. These results support an atomic structural model, based on molecular dynamics simulations and mutagenesis studies, in which the PLB pentamer is stabilized by a leucine-isoleucine zipper within the transmembrane domain.
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PMID:Direct spectroscopic detection of molecular dynamics and interactions of the calcium pump and phospholamban. 1060 46

One of the putative actin-binding sites of Dictyostelium myosin II is the beta-strand-turn-beta-strand structure (Ile(398)-Leu-Ala-Gly-Arg-Asp(403)-Leu-Val(405)), the "myopathy loop, " which is located at the distal end of the upper 50-kDa subdomain and next to the conserved arginine (Arg(397)), whose mutation in human cardiac myosin results in familial hypertrophic cardiomyopathy. The myopathy loop contains the TEDS residue (Asp(403)), which is a target of the heavy-chain kinase in myosin I. Moreover, the loop contains a cluster of hydrophobic residues (Ile(398), Leu(399), Leu(404), and Val(405)), whose side chains are fully exposed to the solvent. In our study, the myopathy loop was deleted from Dictyostelium myosin II to investigate its functional roles. The mutation abolished hydrophobic interactions of actin and myosin in the strong binding state during the ATPase cycle. Association of the mutant myosin and actin was maintained only through ionic interactions under these conditions. Without strong hydrophobic interactions, the mutant myosin still exhibited motor functions, although at low levels. It is likely that the observed defects resulted mainly from a loss of the cluster of hydrophobic residues, since replacement of Asp(403) or Arg(402) with alanine generated a mutant with less severe or no defects compared with those of the deletion mutant.
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PMID:Deletion of the myopathy loop of Dictyostelium myosin II and its impact on motor functions. 1060 48

DNA-gyrase exhibits an unusual ATP-binding site that is formed as a result of gyrase B subunit dimerization, a structural transition that is also essential for DNA capture during the topoisomerization cycle. Previous structural studies on Escherichia coli DNA-gyrase B revealed that dimerization is the result of a polypeptidic exchange involving the N-terminal 14 amino acids. To provide experimental data that dimerization is critical for ATPase activity and enzyme turnover, we generated mutants with reduced dimerization by mutating the two most conserved residues of the GyrB N-terminal arm (Tyr-5 and Ile-10 residues). Our data demonstrate that the hydrophobic Ile-10 residue plays an important role in enzyme dimerization and the nucleotide-protein contact mediated by Tyr-5 side chain residue helps the dimerization process. Analysis of ATPase activities of mutant proteins provides evidence that dimerization enhances the ATP-hydrolysis turnover. The structure of the Y5S mutant of the N-terminal 43-kDa fragment of E. coli DNA GyrB subunit indicates that Tyr-5 residue provides a scaffold for the ATP-hydrolysis center. We describe a channel formed at the dimer interface that provides a structural mechanism to allow reactive water molecules to access the gamma-phosphate group of the bound ATP molecule. Together, these results demonstrate that dimerization strongly contributes to the folding and stability of the catalytic site for ATP hydrolysis. A role for the essential Mg(2+) ion for the orientation of the phosphate groups of the bound nucleotide inside the reactive pocket was also uncovered by superposition of the 5'-adenylyl beta-gamma-imidodiphosphate (ADPNP) wild-type structure to the salt-free ADPNP structure.
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PMID:Dimerization of Escherichia coli DNA-gyrase B provides a structural mechanism for activating the ATPase catalytic center. 1073 94

In the P(2)-type ATPases, there is growing evidence that four alpha-helical stalk segments connect the cytoplasmic part of the molecule, responsible for ATP binding and hydrolysis, to the membrane-embedded part that mediates cation transport. The present study has focused on stalk segment 4, which displays a significant degree of sequence conservation among P(2)-ATPases. When site-directed mutants in this region of the yeast plasma membrane H(+)-ATPase were constructed and expressed in secretory vesicles, more than half of the amino acid substitutions led to a severalfold decrease in the rate of ATP hydrolysis, although they had little or no effect on the coupling between hydrolysis and transport. Strikingly, mutant ATPases bearing single substitutions of 13 consecutive residues from Ile-359 through Gly-371 were highly resistant to inorganic orthovanadate, with IC(50) values at least 10-fold above those seen in the wild-type enzyme. Most of the same mutants also displayed a significant reduction in the K(m) for MgATP and an increase in the pH optimum for ATP hydrolysis. Taken together, these changes in kinetic behavior point to a shift in equilibrium from the E(2) conformation of the ATPase toward the E(1) conformation. The residues from Ile-359 through Gly-371 would occupy three full turns of an alpha-helix, suggesting that this portion of stalk segment 4 may provide a conformationally active link between catalytic sites in the cytoplasm and cation-binding sites in the membrane.
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PMID:Stalk segment 4 of the yeast plasma membrane H+-ATPase. Mutational evidence for a role in the E1-E2 conformational change. 1079 59

By means of a functional expression system and site-directed mutagenesis, we analyzed the role of the putative K(+)-binding site, Glu-345, located in the fourth transmembrane segment of the gastric H(+),K(+)-ATPase alpha-subunit. In the present study, we used several mutants, with alanine, isoleucine, leucine, glutamine, valine, lysine, and aspartic acid instead of Glu-345, and analyzed the H(+),K(+)-ATPase partial reactions of the mutants to determine the precise role of this residue. All the mutants except E345Q exhibited no H(+),K(+)-ATPase activity. The E345Q mutant showed 3-times higher affinity for ATP. This mutation shifted the optimum pH toward a more alkaline one. The E345A, E345I, E345L, E345V as well as E345Q mutants were phosphorylated with ATP as in the case of the wild-type H(+),K(+)-ATPase, whereas the E345K mutant was not phosphorylated. The E345Q mutant was dephosphorylated in the presence of K(+), but its affinity for K(+) was significantly lower than that of the wild type. The E345A, E345I, E345L, and E345V mutants did not exhibit sensitivity to K(+) in the dephosphorylation step below 3 mM K(+). Therefore, Glu-345 is important for the conformational change induced by K(+), especially in the dephosphorylation step in which K(+) reacts with the enzyme from the luminal side with high affinity and accelerates the release of inorganic phosphate. The glutamic acid in the fourth transmembrane segment is conserved, and was found to be involved in the cation-induced conformational change in H(+),K(+)-ATPase as well as Na(+),K(+)-ATPase and Ca(2+)-ATPase, however, the precise roles of the side chain in the function were different.
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PMID:Mutational analysis of the putative K(+)-binding site on the fourth transmembrane segment of the gastric H(+),K(+)-ATPase. 1083 67

Sixteen residues in stalk segment S5 of the Ca(2+)-ATPase of sarcoplasmic reticulum were studied by site-directed mutagenesis. The rate of the Ca(2+) binding transition, determined at 0 degrees C, was enhanced relative to wild type in mutants Ile(743) --> Ala, Val(747) --> Ala, Glu(748) --> Ala, Glu(749) --> Ala, Met(757) --> Gly, and Gln(759) --> Ala and reduced in mutants Asp(737) --> Ala, Asp(738) --> Ala, Ala(752) --> Leu, and Tyr(754) --> Ala. In mutant Arg(762) --> Ile, the rate of the Ca(2+) binding transition was wild type like at 0 degrees C, whereas it was 3.5-fold reduced relative to wild type at 25 degrees C. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was increased conspicuously in mutants Ile(743) --> Ala and Tyr(754) --> Ala (close to 20-fold in the absence of K(+)) and increased to a lesser extent in Asn(739) --> Ala, Glu(749) --> Ala, Gly(750) --> Ala, Ala(752) --> Gly, Met(757) --> Gly, and Arg(762) --> Ile, whereas it was reduced in mutants Asp(737) --> Ala, Val(744) --> Gly, Val(744) --> Ala, Val(747) --> Ala, and Ala(752) --> Leu. In mutants Ile(743) --> Ala, Tyr(754) --> Ala, and Arg(762) --> Ile, the apparent affinities for vanadate were enhanced 23-, 30-, and 18-fold, respectively, relative to wild type. The rate of Ca(2+) dissociation was 11-fold increased in Gly(750) --> Ala and 2-fold reduced in Val(747) --> Ala. Mutants with alterations to Arg(751) either were not expressed at a significant level or were completely nonfunctional. The findings show that S5 plays a crucial role in mediating communication between the Ca(2+) binding pocket and the catalytic domain and that Arg(751) is important for both structural and functional integrity of the enzyme.
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PMID:Importance of stalk segment S5 for intramolecular communication in the sarcoplasmic reticulum Ca2+-ATPase. 1087 20

UV irradiation of the sarcoplasmic reticulum (SR) ATPase in the presence of vanadate cleaves the enzyme at either of two different sites. Under conditions favoring the presence of monovanadate, and in the presence of Ca(2+), ADP, and Mg(2+), cleavage results in two fragments of 71- and 38-kDa electrophoretic mobility. On the other hand, under conditions permitting formation of decavanadate, and in the absence of Ca(2+) and ADP, cleavage results in two fragments of 88- and 21-kDa electrophoretic mobility. The amino terminus resulting from cleavage is blocked and resistant to Edman degradation. However, the initial photo-oxidation product can be reduced with NaB(3)H(4,) resulting in incorporation of radioactive (3)H label. Extensive digestion of the labeled protein with trypsin then yields labeled peptides that are specific for the each of the photo-oxidation conditions, and can be sequenced after purification. Collection of the Edman reaction fractional products reveals the radioactive label and demonstrates that Thr(353) is the residue oxidized by monovanadate at the phosphorylation site (i.e. Asp(351)). Correct positioning of monovanadate at the phosphorylation site requires binding of Mg(2+) and ADP to the Ca(2+)-dependent conformation of the enzyme. Subsequent hydrolytic cleavage is likely assisted by the neighboring Asp(601), and yields the 71- and 38-kDa fragments. On the other hand, Ser(186) (and possibly the following three residues: Val(187), Ile(188), and Lys(189)) is the residue that is photo-oxidized by decavanadate in the absence of ADP. Hydrolytic cleavage of the oxidized product at this site is likely assisted by neighboring acidic residues, and yields the 88- and 21-kDa fragments. The bound decavanadate, which we find to produce steric interference with TNP-AMP binding, must therefore extend to the A domain (i.e. small cytosolic loop) in order to oxidize Ser(186). This protein conformation is only obtained in the absence of Ca(2+).
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PMID:Distinct topologies of mono- and decavanadate binding and photo-oxidative cleavage in the sarcoplasmic reticulum ATPase. 1090 27

Two monoclonal antibodies, beta 208 and beta 210, against the beta subunit of the F(1) ATPase from Escherichia coli reacted with an intact beta subunit and also a peptide corresponding to a portion of beta between residues 1 and 145. Mutations at Ala-1, Val-15, Glu-16, Phe-17, Leu-29, Gly-65, or Leu-66, and His-110 or Arg-111 for beta 210 and beta 208, respectively, caused decreased antibody binding to beta, suggesting that these residues form the epitopes and are thought to lie close together on the surface of the beta subunit. The topological locations of the corresponding residues in the atomic structure of the bovine beta subunit agree well with these expectations, except for Ala-1 and Leu-29. beta 210 binds to two beta strands including the epitope residues that are 50 residues apart, indicating that this antibody recognizes the tertiary structure of the N-terminal end region. Mutations in the epitope residues of beta 210 do not affect the F(1) ATPase activity, suggesting that surfaces of the two beta strands in the amino-terminal end region are not functionally essential. To analyze the functional importance around His-110 recognized by beta 208 we introduced site specific mutations at residues His-110 and Ile-109. Ile-109 to Ala or Arg, and His-110 to Ala or Asp caused defective assembly of F(1). However, the His-110 to Arg mutation had no effect on molecular assembly, suggesting that Ile-109 and His-110, especially the positive charge of His-110 are essential for the assembly of F(1). The His-110 to Arg mutation caused a large decrease in F(1)-ATPase activity, suggesting that a subtle change in the topological arrangement of the positive charge of His-110 located on the surface of beta plays an important role in the catalytic mechanism of the F(1)-ATPase.
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PMID:Monoclonal antibodies recognizing surface residues of the beta subunit of Escherichia coli F(1) ATPase: functional importance of the epitope residues. 1101 Nov 45

Chloroplast ATP synthase is a thiol-modulated enzyme whose DeltamuH(+)-linked activation is strongly influenced by reduction and the formation of a disulphide bridge between Cys(199) and Cys(205) on the gamma subunit. In solubilized chloroplast coupling factor 1 (CF(1)), reduction of the disulphide bond elicits the latent ATP-hydrolysing activity. To assess the regulatory importance of the amino acid residues around these cysteine residues, we focused on the three negatively charged residues Glu(210)-Asp-Glu(212) close to the two cysteine residues and also on the following region from Leu(213) to Ile(230), and investigated the modulation of ATPase activity by chloroplast thioredoxins. The mutant gamma subunits were reconstituted with the alpha and beta subunits from F(1) of the thermophilic bacterium Bacillus PS3; the active ATPase complexes obtained were purified by gel-filtration chromatography. The complex formed with a mutant gamma subunit in which Glu(210) to Glu(212) had been deleted was inactivated rather than activated by reduction of the disulphide bridge by reduced thioredoxin, indicating inverse regulation. This complex was insensitive to the inhibitory CF(1)-epsilon subunit when the mutant gamma subunit was oxidized. In contrast, the deletion of Glu(212) to Ile(230) converted the complex from a modulated state into a highly active state.
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PMID:Inverse regulation of F1-ATPase activity by a mutation at the regulatory region on the gamma subunit of chloroplast ATP synthase. 1110 86


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