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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)

The inhibitory effect of epsilon subunit in F(1)-ATPase from thermophilic Bacillus PS3 was examined focusing on the structure-function relationship. For this purpose, we designed a mutant for epsilon subunit similar to the one constructed by Schulenberg and Capaldi (Schulenberg, B., and Capaldi, R. A. (1999) J. Biol. Chem. 274, 28351-28355). We introduced two cysteine residues at the interface of N-terminal beta-sandwich domain (S48C) and C-terminal alpha-helical domain (N125C) of epsilon subunit. The alpha(3)beta(3)gammaepsilon complex containing the reduced form of this mutant epsilon subunit showed suppressed ATPase activity and gradual activation during the measurement. This activation pattern was similar to the complex with the wild type epsilon subunit. The conformation of the mutant epsilon subunit must be fixed and similar to the reported three-dimensional structure of the isolated epsilon subunit, when the intramolecular disulfide bridge was formed on this subunit by oxidation. This oxidized mutant epsilon subunit could form the alpha(3)beta(3)gammaepsilon complex but did not show any inhibitory effect. The complex was converted to the activated state, and the cross-link in the mutant epsilon subunit in the complex was efficiently formed in the presence of ATP-Mg, whereas no cross-link was observed without ATP-Mg, suggesting the conformation of the oxidized mutant epsilon subunit must be similar to that in the activated state complex. A non-hydrolyzable analog of ATP, 5'-adenylyl-beta,gamma-imidodiphosphate, could stimulate the formation of the cross-link on the epsilon subunit. Furthermore, the cross-link formation was stimulated by nucleotides even when this mutant epsilon subunit was assembled with a mutant alpha(3)beta(3)gamma complex lacking non-catalytic sites. These results indicate that binding of ATP to the catalytic sites induces a conformational change in the epsilon subunit and triggers transition of the complex from the suppressed state to the activated state.
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PMID:Movement of the helical domain of the epsilon subunit is required for the activation of thermophilic F1-ATPase. 1095 1

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

The structure of the F(1)-ATPase from spinach chloroplasts was determined to 3.2 A resolution by molecular replacement based on the homologous structure of the bovine mitochondrial enzyme. The crystallized complex contains four different subunits in a stoichiometry of alpha(3)beta(3)gammaepsilon. Subunit delta was removed before crystallization to improve the diffraction of the crystals. The overall structure of the noncatalytic alpha-subunits and the catalytic beta-subunits is highly similar to those of the mitochondrial and thermophilic subunits. However, in the crystal structure of the chloroplast enzyme, all alpha- and beta-subunits adopt a closed conformation and appear to contain no bound adenine nucleotides. The superimposed crystallographic symmetry in the space group R32 impaired an exact tracing of the gamma- and epsilon-subunits in the complex. However, clear electron density was present at the core of the alpha(3)beta(3)-subcomplex, which probably represents the C-terminal domain of the gamma-subunit. The structure of the spinach chloroplast F(1) has a potential binding site for the phytotoxin, tentoxin, at the alphabeta-interface near betaAsp(83) and an insertion from betaGly(56)-Asn(60) in the N-terminal beta-barrel domain probably increases the thermal stability of the complex. The structure probably represents an inactive latent state of the ATPase, which is unique to chloroplast and cyanobacterial enzymes.
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PMID:The structure of the chloroplast F1-ATPase at 3.2 A resolution. 1103 39

Since the chemiosmotic theory was proposed by Peter Mitchell in the 1960s, a major objective has been to elucidate the mechanism of coupling of the transmembrane proton motive force, created by respiration or photosynthesis, to the synthesis of ATP from ADP and inorganic phosphate. Recently, significant progress has been made towards establishing the complete structure of ATP synthase and revealing its mechanism. The X-ray structure of the F(1) catalytic domain has been completed and an electron density map of the F(1)-c(10) subcomplex has provided a glimpse of the motor in the membrane domain. Direct microscopic observation of rotation has been extended to F(1)-ATPase and F(1)F(o)-ATPase complexes.
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PMID:The rotary mechanism of ATP synthase. 1111 4

A soluble F(1)-ATPase was isolated from the mitochondria of crayfish (Orconectes virilis) gill tissue. The maximal mitochondrial disruption rate (95%) was obtained by sonicating for 4 min at pH 8.6. A 15-fold purification was estimated. The properties for both soluble and membrane-bound enzyme were studied. Both enzyme forms were stable at 4 to -70 degrees C when kept in 20% glycerol. Soluble F(1)-ATPase was more stable at room temperature than membrane-bound enzyme. It displayed a narrower pH profile (pK(1) =6.58, pK(2)=7.68) and more acid pH optimum (7.13) than membrane-bound enzyme (pK(1)=6.42, pK(2)=8.55, optimum pH 7.49). The anion-stimulated activities were in the order HCO(3)(-)>SO(4)(2-)>Cl(-). The apparent K(a) values for soluble enzyme were 11.4, 11.2, and 10.9 mM, respectively, but the K(a) of HCO(3)(-) for membrane-bound enzyme (14.9 mM) was higher than for soluble enzyme. Oligomycin and DCCD inhibited membrane-bound F(1)-ATPase with I(50) of 18.6 ng/ml and 2.2 microM, respectively, but were ineffective in inhibiting soluble enzyme. Both enzyme forms shared identical sensitivity to DIDS (I(50)=12.5 microM) and vanadate (I(50)=9.0 mM). Soluble ATPase was significantly more sensitive to pCMB (I(50)=0.15 microM) and NO(3)(-) (I(50)=28.6 mM) than membrane-bound enzyme (I(50)=1.04 microM pCMB and 81.5 mM NO(3)(-)). In addition, soluble F(1)-ATPase was slightly more sensitive to azide (I(50)=91.8 microM) and NBD-Cl (I(50)=9.18 microM) than membrane-bound enzyme (I(50)=111.6 microM azide and 12.88 microM NBD-Cl). These data suggest a conformational change transmission between F(0) and F(1) sectors and slight conformational differences between soluble F(1) and membrane-bound F(1). In addition, an unmodified F(0) stabilizes F(1) and decreases F(1) sensitivities to inhibitors and modulators.
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PMID:Isolation and characterization of mitochondrial F(1)-ATPase from crayfish (Orconectes virilis) gills. 1120 45

The substrate kinetics and the role of free Mg(2+) and free ATP were studied in membrane-bound F(1)-ATPase from crayfish (Orconectes virilis) gills. It was shown that the MgATP complex was the true substrate for the ATPase activity with a K(m) value of 0.327 mM. In the absence of bicarbonate, the maximum azide-sensitive activities in the presence and absence (<18 microM) of free ATP were 0.878 and 0.520 micromol P(i)/mg protein/min, respectively, while the maximum bicarbonate-stimulated activity in absence of free ATP was 1.486 micromol P(i)/mg protein/min. Free ATP was a competitive inhibitor (K(i)=0.77 mM) and free Mg(2+) was a mixed inhibitor (K(i)=0.81 mM, K(i)'=5.89 mM). However, free ATP also acted as an activator. Lineweaver-Burk plots for MgATP hydrolysis at high free Mg(2+) concentrations exhibited an apparent negative cooperativity, which was not the case for high free ATP levels. These results suggest that, although free ATP inhibited the enzyme by binding to catalytic sites, it stimulated ATPase activity by binding to non-catalytic sites and promoted the dissociation of inhibitory MgADP from the catalytic site.
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PMID:Kinetic studies on mitochondrial F(1)-ATPase from crayfish (Orconectes virilis) gills. 1120 46

In F(1)-ATPase, a rotary motor enzyme, the region of the conserved DELSEED motif in the beta subunit moves and contacts the rotor gamma subunit when the nucleotide fills the catalytic site, and the acidic nature of the motif was previously assumed to play a critical role in rotation. Our previous work, however, disproved the assumption (Hara, K. Y., Noji, H., Bald, D., Yasuda, R., Kinosita, K., Jr., and Yoshida, M. (2000) J. Biol. Chem. 275, 14260-14263), and the role of this motif remained unknown. Here, we found that the epsilon subunit, an intrinsic inhibitor, was unable to inhibit the ATPase activity of a mutant thermophilic F(1)-ATPase in which all of the five acidic residues in the DELSEED motif were replaced with alanines, although the epsilon subunit in the mutant F(1)-ATPase assumed the inhibitory form. In addition, the replacement of basic residues in the C-terminal region of the epsilon subunit by alanines caused a decrease of the inhibitory effect. Partial replacement of the acidic residues in the DELSEED motif of the beta subunit or of the basic residues in the C-terminal alpha-helix of the epsilon subunit induced a partial effect. We here conclude that the epsilon subunit exerts its inhibitory effect through the electrostatic interaction with the DELSEED motif of the beta subunit.
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PMID:The role of the betaDELSEED motif of F1-ATPase: propagation of the inhibitory effect of the epsilon subunit. 1127 33

Site-directed mutations were made to the phosphate-binding loop lysine in the beta-subunit of the chloroplast F(1)-ATPase in Chlamydomonas reinhardtii (betaK167) to investigate the participation of this residue in the binding of metal to catalytic site 3 in the absence of nucleotide. The cw-EPR spectra of VO(2+) bound to site 3 of CF(1)-ATPase from wild type and mutants revealed changes in metal ligation resulting from mutations to betaK167. The three-pulse ESEEM spectrum of the wild-type CF(1)-ATPase with VO(2+) bound to site 3 shows an equatorially coordinating (14)N from an amine. The ESEEM spectra of the mutants do not show evidence of an equatorially coordinating amine group. The results presented here show that, in the absence of nucleotide, betaK167 is a ligand to the metal bound at catalytic site 3, suggesting a regulatory role for the P-loop lysine in addition to its known role in catalysis.
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PMID:Identification of the P-loop lysine as a metal ligand in the absence of nucleotide at catalytic site 3 of chloroplast F1-ATPase from Chlamydomonas reinhardtii. 1129 39

Site-directed mutants Y317C, Y317E, Y317F, Y317G, and Y317K were made to the catch-loop tyrosine on the beta subunit of the chloroplast F(1)-ATPase in Chlamydomonas. EPR spectra of VO(2+)-ATP bound to site 3 of CF(1) from wild type and mutants were obtained. Every mutant changed the (51)V hyperfine parameters of the VO(2+) bound at this site in the catalytically active conformation of the enzyme but had no effect on these parameters in the form that predominates when the enzyme activity is latent. These results indicate that this residue is a ligand to the metal of the Mg(2+)-nucleotide complex that binds to the empty catalytic site. The mutations also decreased the k(cat) of the ATPase activity to a much greater extent than k(cat)/K(M). Thus, these mutations limit the rate of product (Mg(2+)-ADP and phosphate) release in the ATPase direction or, conversely, the initial binding of substrates in the ATP synthesis direction. On the basis of these observations, coordination of betaY317 by Mg(2+)-ADP that binds to the empty catalytic site provides a means by which substrate binding could trigger gamma subunit rotation and consequent conformation changes of beta subunits during ATP synthesis.
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PMID:Interaction of the catch-loop tyrosine beta Y317 with the metal at catalytic site 3 of Chlamydomonas chloroplast F1-ATPase. 1141 27

In F(1)-ATPase, the smallest known motor enzyme, unidirectional rotation of the central axis subunit gamma is coupled to ATP hydrolysis. In the present study, we report the redox switching of the rotation of this enzyme. For this purpose, the switch region from the gamma subunit of the redox-sensitive chloroplast F(1)-ATPase was introduced into the bacterial F(1)-ATPase. The ATPase activity of the obtained complex was increased up to 3-fold upon reduction (Bald, D., Noji, H., Stumpp, M. T., Yoshida, M. & Hisabori, T. (2000) J. Biol. Chem. 275, 12757-12762). Here, we successfully observed the modulation of rotation of gamma in this chimeric complex by changes in the redox conditions. In addition we revealed that the suppressed enzymatic activity of the oxidized F(1)-ATPase complex was characterized by more frequent long pauses in the rotation of the gamma subunit. These findings obtained by the single molecule analysis therefore provide new insights into the mechanisms of enzyme regulation.
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PMID:Redox regulation of the rotation of F(1)-ATP synthase. 1151


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