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)

A mutant of Escherichia coli F1-ATPase (ECF1) in which the serine residue in position 8 of the gamma subunit has been replaced by a cysteine residue (gamma S8C) has been used to study nucleotide-dependent cross-linking of the gamma subunit to a beta subunit. When examined in the presence of ADP+Mg2+, either supplied directly or as produced during catalytic turnover of ATP+Mg2+, the main cross-linked product generated using the heterobifunctional, photoactivatable, cross-linker tetrafluorophenylazide maleimide-6 had a M(r)(app) of 108,000. When ATP hydrolysis was inhibited, either by cold or by reaction with sodium azide, or when ATP hydrolysis was prevented by the use of adenyl-5'-yl beta,gamma-imidodiphosphate, the main cross-linked products were species with M(r)(app) of 102,000 and 84,000. The nucleotide-dependent switching from one cross-linking pattern to another could only be observed when the epsilon subunit was bound to ECF1; it was not seen in ECF1*, an enzyme preparation missing delta and epsilon subunits, but was observed in preparations selectively depleted of the delta subunit. We conclude that the changes detected in these cross-linking experiments are occurring during the hydrolysis of ATP when the beta-gamma phosphate bond is cleaved and that they are related to the coupling of ATP hydrolysis to proton translocation.
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PMID:ATP hydrolysis-linked structural changes in the N-terminal part of the gamma subunit of Escherichia coli F1-ATPase examined by cross-linking studies. 839 54

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

The soluble portion of the Escherichia coli F1F0 ATP synthase (ECF1) and E. coli F1F0 ATP synthase (ECF1F0) have been isolated from a novel mutant gammaY205C. ECF1 isolated from this mutant had an ATPase activity 3.5-fold higher than that of wild-type enzyme and could be activated further by maleimide modification of the introduced cysteine. This effect was not seen in ECF1F0. The mutation partly disrupts the F1 to F0 interaction, as indicated by a reduced efficiency of proton pumping. ECF1 containing the mutation gammaY205C was bound to the membrane-bound portion of the E. coli F1F0 ATP synthase (ECF0) isolated from mutants cA39C, cQ42C, cP43C, and cD44C to reconstitute hybrid enzymes. Cu2+ treatment or reaction with 5,5'-dithio-bis(2-nitro-benzoic acid) induced disulfide bond formation between the Cys at gamma position 205 and a Cys residue at positions 42, 43, or 44 in the c subunit but not at position 39. Using Cu2+ treatment, this covalent cross-linking was obtained in yields as high as 95% in the hybrid ECF1 gammaY205C/cQ42C and in ECF1F0 isolated from the double mutant of the same composition. The covalent linkage of the gamma to a c subunit had little effect on ATPase activity. However, ATP hydrolysis-linked proton translocation was lost, by modification of both gamma Cys-205 and c Cys-42 by bulky reagents such as 5,5'-dithio-bis (2-nitro-benzoic acid) or benzophenone-4-maleimide. In both ECF1 and ECF1F0 containing a Cys at gamma 205 and a Cys in the epsilon subunit (at position 38 or 43), cross-linking of the gamma to the epsilon subunit was induced in high yield by Cu2+. No cross-linking was observed in hybrid enzymes in which the Cys was at position 10, 65, or 108 of the epsilon subunit. Cross-linking of gamma to epsilon had only a minimal effect on ATP hydrolysis. The reactivity of the Cys at gamma 205 showed a nucleotide dependence of reactivity to maleimides in both ECF1 and ECF1F0, which was lost in ECF1 when the epsilon subunit was removed. Our results show that there is close interaction of the gamma and epsilon subunits for the full-length of the stalk region in ECF1F0. We argue that this interaction controls the coupling between nucleotide binding sites and the proton channel in ECF1F0.
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PMID:The stalk region of the Escherichia coli ATP synthase. Tyrosine 205 of the gamma subunit is in the interface between the F1 and F0 parts and can interact with both the epsilon and c oligomer. 891 Apr 57

Glutamic acid-190 in the beta subunit of F1-ATPase from thermophilic Bacillus PS-3 (TF1) was reported to be essential for the ATPase activity. The mutant TF1beta subunit in which Glu-190 had been substituted by cysteine was carboxymethylated with 13C-labeled monoiodoacetic acid. The pKa value of the carboxymethylene group at the 190 position was determined as 5.6 +/- 0.4 by 13C-NMR. On the basis of this value, the pKa of the carboxylate of Glu-190 of the TF1beta subunit was estimated to be 6.8 +/- 0.5. The unusually high pKa could play a role in the catalytic mechanism of F1-ATPase.
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PMID:Unusual pKa of the carboxylate at the putative catalytic position of the thermophilic F1-ATPase beta subunit determined by 13C-NMR. 894 27

Ceroid lipofuscinosis protein (CLP), the major accumulating protein in several forms of ceroid lipofuscinosis, has an amino acid sequence that is identical to that of the F0 subunit c of normal bovine ATP synthase. Electrospray ionization mass spectrometry (ESI-MS) has shown that ovine CLP and normal bovine F0 subunit c are identical, including a 42 mass unit post-translational modification. Although the identity and the location of this modification have not been fully established in both species, CLP can be used as a convenient and a unique source of subunit c for studies of F0 inhibitor interactions by ESI-MS analysis. Analysis of mixtures of CLP incubated with several known F0 inhibitors showed that N, N'-dicyclohexylcarbodiimide and organotins bind covalently to CLP but interactions with oligomycin and venturicidin were not observed. The sulphydryl inhibitors, 2,3-dimethoxy-5-methyl-1,4,-benzoquinone (UQ0) and N-ethyl maleimide (NEM) were also shown to bind covalently to the protein. The binding stoichiometry and the relative rate of reaction were then determined for each inhibitor. Tandem mass spectrometry experiments performed on the [M+5H]5+ ion of the intact CLP and of the complexes UQ0-CLP and NEM-CLP allowed the identification of 80% of the CLP sequence and revealed that UQ0 and NEM are both bound to cysteine-64. This work shows the exceptional utility of ESI-MS in studies of the interaction of CLP with a range of inhibitors which are applicable to studies of the F0 component of ATP synthase.
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PMID:Use of electrospray ionization mass spectrometry and tandem mass spectrometry to study binding of F0 inhibitors to ceroid lipofuscinosis protein, a model system for subunit c of mitochondrial ATP synthase. 901 34

The N-ethylmaleimide reactivity of c subunits in Escherichia coli F1F0 ATP synthase (ECF1F0) isolated from five mutants, each with a cysteine at a different position in the polar loop region (positions 39, 40, 42, 43, and 44), has been investigated. The maleimide was found to react with Cys placed at positions 42, 43, and 44 but not at 39 or 40. All copies of the c subunit reacted similarly when the Cys was at position 43 or 44. In contrast, the Cys in the mutant cQ42C reacted as two classes, with 60% reacting relatively rapidly and 40% reacting at a rate 40-fold slower. After removing F1, all copies of the c subunit in this mutant reacted equally fast. Therefore, the slow class in the cQ42C mutant represents c subunits shielded by, and probably involved directly in, the interaction of the F0 with gamma and epsilon subunits of the F1 part. Based on the estimated stoichiometry of c subunits in the ECF1F0 complex, 4 or 5 c subunits are involved in this F1 interaction. N-Ethylmaleimide modification of all of the c subunits reduced ATPase activity by only 30% in ECF1F0 from mutant cQ42C. Modification of the more rapidly reacting class had little effect on ATP hydrolysis-driven proton translocation, and did not alter the DCCD inhibition of ATPase activity. However, as those c subunits involved in the F1 interaction became modified, DCCD inhibition was progressively lost, as was coupling between ATP hydrolysis and proton translocation.
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PMID:Interactions between the F1 and F0 parts in the Escherichia coli ATP synthase. Associations involving the loop region of C subunits. 918 24

Previously, we reported the substitution of Tyr341 of the F1-ATPase beta subunit from a thermophilic Bacillus strain PS3 with leucine, cysteine, or alanine (M. Odaka et al. J. Biochem., 115 (1994) 789-796). These mutations resulted in a great decrease in the affinity of the isolated beta subunit for ATP-Mg and an increase in the apparent Km of the alpha3beta3gamma complex in ATP hydrolysis when examined above 0.1 mM ATP. Here, we examined the ATPase activity of the mutant complexes in a wide range of ATP concentration and found that the mutants exhibited apparent positive cooperativity in ATP hydrolysis. This is the first clear demonstration that a single mutation in the catalytic sites converts the kinetics from apparent negative cooperativity in the wild-type alpha3beta3gamma complex to apparent positive cooperativity. The conversion of apparent cooperativity could be explained in terms of a simple kinetic scheme based on the binding change model proposed by Boyer.
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PMID:A single mutation at the catalytic site of TF1-alpha3beta3gamma complex switches the kinetics of ATP hydrolysis from negative to positive cooperativity. 928 16

All 21 native cysteines in the Escherichia coli F(0)F1 ATP synthase were replaced by alanines. In isolated E. coli membranes, ATP-dependent proton pumping, turnover of ATP hydrolysis and steady-state transition state thermodynamic parameters of the cysteine-less enzyme were similar to wild-type. The cysteine-less enzyme was solubilized in n-octyl beta-D-glucopyranoside, purified by affinity chromatography, and reconstituted into pre-formed liposomes made from E. coli lipids. The properties of the reconstituted, purified enzyme were not significantly different from the membranous enzyme. These data demonstrate that cysteine-less F(0)F1 is biochemically stable and has functionality similar to wild-type.
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PMID:Stability and functionality of cysteine-less F(0)F1 ATP synthase from Escherichia coli. 959 11

An affinity resin for the F1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b24-156, a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F1. Truncated forms of b24-156, in which one or four residues from the C terminus were removed, competed poorly for F1 binding, suggesting that these residues play an important role in b-F1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b24-156 resulted in a disruption of its association with the purified delta subunit of the enzyme. To determine whether these residues interact directly with delta, cysteine residues were introduced at various C-terminal positions of b and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and delta were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b24-156-F1 complex and the membrane-bound F1F0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue delta subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that the b and delta subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F1 sector to the b subunit of F0.
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PMID:The b and delta subunits of the Escherichia coli ATP synthase interact via residues in their C-terminal regions. 961 29

Subunit a of the E. coli F1F0 ATP synthase was probed by insertion scanning mutagenesis in a region between residues Glu219 and His245. A series of single amino acid insertions, of both alanine and aspartic acid, were constructed after the following residues: 225, 229, 233, 238, 243, and 245. The mutants were tested for growth yield, binding of F1 to membranes, dicyclohexylcarbodiimide sensitivity of ATPase activity, ATP-driven proton translocation, and passive proton permeability of membranes stripped of F1. Significant loss of function was seen only with insertions after positions 238 and 243. In contrast, both insertions after residue 225 and the alanine insertion after residue 245 were nearly identical in function to the wild type. The other insertions showed an intermediate loss of function. Missense mutations of His245 to serine and cysteine were nonfunctional, while the W241C mutant showed nearly normal ATPase function. Replacement of Leu162 by histidine failed to suppress the 245 mutants, but chemical rescue of H245S was partially successful using acetate. An interaction between Trp241 and His245 may be involved in gating a "half-channel" from the periplasmic surface of F0 to Asp61 of subunit a.
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PMID:Insertion scanning mutagenesis of subunit a of the F1F0 ATP synthase near His245 and implications on gating of the proton channel. 963 81


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