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)

The incubation of chloroplast thylakoids with pyridoxal 5'-phosphate for a short time (5 s) modified the lysine residues of the ATP synthase beta subunit. Except for lysine residues in the N-terminal and C-terminal regions, the glycine-rich P-loop (GGAGVGKT)-, Lys154-, and Lys167-containing peptide (P-peptide) exhibited high reactivity with pyridoxal 5'-phosphate. The energization of thylakoids or addition of substrates (ADP, Pi, ATP) affected the modifications of the P-peptide, Lys447, and Lys399. For the P-peptide, substrates inhibited the modification with 0.5 mM ADP inhibiting by 80%. Energization enhanced the inhibitory effects of substrates. For Lys447, substrates also inhibited the modification; 0.5 mM ADP inhibited by 60%. For Lys399, the reactivity depended on the transmembrane delta mu H+. With increasing delta mu H+, the reactivity decreased. These results suggest the existence of energy-dependent conformational changes at the catalytic nucleotide-binding site and around Lys399. The former increases the affinity of the site for substrates. Substrate binding at the catalytic site changes the conformation around Lys447.
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PMID:Effects of energization and substrates on the reactivities of lysine residues of the chloroplast ATP synthase beta subunit. 770 38

A role in coupling proton transport to catalysis of ATP synthesis has been demonstrated for the Escherichia coli F0F1 ATP synthase gamma subunit. Previously, functional interactions between the terminal regions that were important for coupling were shown by finding several mutations in the carboxyl-terminal region of the gamma subunit (involving residues at positions 242 and 269-280) that restored efficient coupling to the mutation, gamma Met-23-->Lys (Nakamoto, R. K., Maeda, M., and Futai, M. (1993) J. Biol. Chem. 268, 867-872). In this study, we used suppressor mutagenesis to establish that the terminal regions can be separated into three interacting segments. Second-site mutations that cause pseudo reversion of the primary mutations, gamma Gln-269-->Glu or gamma Thr-273-->Val, map to an amino-terminal segment with changes at residues 18, 34, and 35, and to a segment near the carboxyl terminus with changes at residues 236, 238, 242, and 246. Each second-site mutation suppressed the effects of both gamma Gln-269-->Glu and gamma Thr-273-->Val, and restored efficient coupling to enzyme complexes containing either of the primary mutations. Mapping of these residues in the recently reported x-ray crystallographic structure of the F1 complex (Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), reveals that the second-site mutations do not directly interact with gamma Gln-269 and gamma Thr-273 and that the effect of suppression occurs at a distance. We propose that the three gamma subunit segments defined by suppressor mutagenesis, residues gamma 18-35, gamma 236-246, and gamma 269-280, constitute a domain that is critical for both catalytic function and energy coupling.
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PMID:The ATP synthase gamma subunit. Suppressor mutagenesis reveals three helical regions involved in energy coupling. 777 64

A molecular genetic approach has been used to test the proposition that the central hydrophobic domain of yeast mitochondrial ATP synthase subunit 8 represents a transmembrane stem in contact with the lipid bilayer. The rationale for this approach is the general inability of membrane bilayers to accomodate unshielded charged residues of polypeptide chains. Non-polar residues at several positions within the central hydrophobic domain of subunit 8 were replaced with the positively charged amino acid lysine. This was done in an attempt to disrupt subunit 8 function, and thereby determine the boundaries of the putative transmembrane stem. Each subunit 8 variant was allotopically expressed in vivo as a mitochondrial import precursor encoded by a nuclear gene. It was found that all variants, which included proteins carrying two lysines at various positions in the hydrophobic domain, exhibited the ability to restore growth of subunit-8-deficient cells on the non-fermentable substrate ethanol. This indicated that the function of none of these subunit 8 variants was severely compromised. There was also no detectable change in the proteolipid characteristics of subunit 8, as defined by the chloroform/methanol solubility properties of variant proteins extracted from membranes following import into isolated mitochondria. These data suggest that subunit 8 is located in a hydrophobic niche in the mitochondrial ATP synthase, probably in contact with other protein subunits of the complex. We conclude that the function of subunit 8 does not necessarily require it to be integrated within the inner mitochondrial membrane, in contact with the lipid bilayer. Our findings also suggest that hydropathy plots, indicating hydrophobic domains within polypeptides, cannot reliably be interpreted as transmembrane helices in the absence of independent evidence.
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PMID:Relationship of subunit 8 of yeast ATP synthase and the inner mitochondrial membrane. Subunit 8 variants containing multiple lysine residues in the central hydrophobic domain retain function. 786 34

The Q42E mutation in the polar loop of subunit c of the Escherichia coli F1F0 ATP synthase leads to an uncoupling of H+ translocation through F0 and ATP synthesis/hydrolysis in F1. We have isolated four second-site suppressor mutants in which the coupling defect is corrected. Substitutions for Glu31 in F1 subunit epsilon were found in each suppressor mutant, where the substitutions were E31G, E31V, and E31K (the last being found twice). The different substitutions vary in effectiveness in restoring wild type growth properties in the order epsilon E31G > epsilon E31V > epsilon E31K. Biochemical properties of epsilon E31G/cQ42E and epsilon E31K/cQ42E membranes were compared. In epsilon E31G/cQ42E mutant membranes, ATP-driven H+ translocation by F1F0 and the binding and coupling of F1 to F0 showed a striking pH dependence. Near normal function was observed at pH 7.0, but function was lost at pH 7.8. The function of epsilon E31K/cQ42E membranes was much less affected by changes in pH. Relative to epsilon E31G/cQ42E membranes, the ATP-driven H+ transport function of epsilon E31K/cQ42E membranes was approximately the same at pH 7.5, greater at pH 7.8, and less at pH 7.0. The differences between mutants could be explained if cGlu42 ionized at pH 7.8 with loss of function in epsilon E31G/cQ42E membrane and a similar ionization were compensated for by the positively charged Lys in the epsilon E31K/cQ42E membrane.
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PMID:Suppressor mutations in F1 subunit epsilon recouple ATP-driven H+ translocation in uncoupled Q42E subunit c mutant of Escherichia coli F1F0 ATP synthase. 790 91

We analyzed reversion mutations of the mutants with a substitution of Leu-40 by Pro or Glu-41 by Lys in the beta subunit of Escherichia coli F1-ATPase. These mutants had an altered molecular assembly of the alpha and beta subunit on the membranes and lost the binding activity of the beta subunit to the monoclonal antibody beta 31. For the mutation of Leu-40 to Pro, we found that all reversion mutations Gln, or Ser besides the wild-type Leu. These pseudo reversion mutations restored the cell growth on minimal agar supplemented with succinate as the sole carbon source, the assembly of the alpha and beta subunits, and also the ATPase activities in the membranes. These results suggested that Leu-40 itself is not an essential residue for the function of the beta subunit but that the residue contributes to a conformation around residue 40, which is important for the assembly of the alpha and beta subunits onto the membranes. For the mutation of Glu-41 to Lys, pseudo reversion mutations were found at the original residue 41 as Lys to Gln or Asn and also at Arg-218 to Cys or His in addition to the original Glu-41 to Lys mutation. These suppression mutations recovered the cell growth, indicating the recovery of ATP synthesis. The ATPase activity was high in cells with the Lys-41 to Glu mutation but those were relatively low in those with other reversion mutations. The assembly of the alpha and beta subunits recovered in the revertants to the wild-type level, except for the Lys-41 to Asn mutation, which resulted in a decreased amount of the alpha subunit on the membranes. These results suggested that though Glu-41 is not an essential residue, it plays an important role in the assembly of the alpha and beta subunit on the membranes. The results also suggested that residues Arg-218 and Glu-41 are located close together and interact with each other, either directly or indirectly.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Reversion mutations in the beta subunit mutants of Escherichia coli F1-ATPase with defective subunit assembly: implications for structure and function of the amino-terminal region. 791 94

The exposure to trypsinolysis of subunits of F1F0-ATPase and of its F0 domain have been compared in everted inner membrane vesicles (submitochondrial particles) made from bovine mitochondria. Treatment of submitochondrial particles with guanidine hydrochloride removed the subunits of F1-ATPase and the oligomycin-sensitivity conferral protein (OSCP), and exposed sites that were occluded in the intact F1F0-ATPase complex. These sites were identified by purifying the subunits from the isolated F0 and F1F0-ATPase complexes before and after proteolysis of the vesicles, and by characterizing them by N-terminal sequencing and electrospray-ionization mass spectrometry. In the stripped vesicles, subunit F6 was completely digested away by either trypsin or chymotrypsin. Trypsin also cleaved subunit b, first at the bond arginine-166-glutamine-167, and then at the consecutive linkages, lysine-120-arginine-121 and arginine-121-histidine-122. Chymotrypsin-sensitive sites were observed after the adjacent methionines 164 and 165. Trypsin also removed amino acids 1-3 of subunit d, and minor cleavage sites were observed in subunit d between amino acids 24 and 25, in subunit g between amino acids 5 and 6, and after amino acid 40 in subunit e. The other subunits remained protected from proteolysis. In membrane-bound F1F0-ATPase, the N-terminus of subunit d was also accessible to trypsin, and subunit e was more susceptible to proteolysis than in F0. Otherwise the F0 subunits and the OSCP were protected. Subunits alpha and beta were cleaved by trypsin at the same sites in their N-terminal regions as in purified F1-ATPase. The trypsinized F0 was incapable of binding F1-ATPase in the presence of the OSCP. These experiments and in vitro re-assembly experiments described elsewehere, that were guided by the results of the proteolysis experiments, have helped to establish a central role for subunit b in the formation of the stalk connecting the F1 and F0 domains of the F1F0-ATPase complex.
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PMID:ATP synthase from bovine heart mitochondria: identification by proteolysis of sites in F0 exposed by removal of F1 and the oligomycin-sensitivity conferral protein. 798 Apr 27

Three amino acid residues in the a subunit of the Escherichia coli F1F0 ATP synthase are essential for proton translocation: Arg210, Glu219, and His245. In this study, the essential glutamic acid has been relocated to position 252 with retention of function. It had been known that Gln252 can be replaced by Glu without significant effect. To test whether Q252E would function in the absence of Glu219, a "site-directed second-site suppressor" experiment was designed. Saturation mutagenesis was applied to residue Glu219, and 14 different amino acid substitutions were isolated, five of which permitted growth on succinate minimal medium at 37 degrees C: Asp, Lys, Gly, Ala, and Ser. These results indicate that Q252E can provide the essential carboxyl group normally provided by Glu219, but that strict requirements are placed on the residue at position 219. We interpret these results to mean that the Q252E must occupy, at least partially, the normal position of Glu219. We present a novel mechanism of proton translocation by F1F0 ATP synthases that includes a rotating oligomer of c subunits, in which the Asp61 of two c subunits simultaneously interact with Glu219 and Arg210 of the a subunit. This mechanism can be adapted for both mitochondrial and sodium-driven bacterial ATP synthases.
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PMID:A mechanism of proton translocation by F1F0 ATP synthases suggested by double mutants of the a subunit. 798 50

Nucleotide binding proteins, including ras, elongation factor Tu, adenylate kinase, and the mitochondrial F1-ATPase have a glycine-rich motif known as the P-loop or the Walker A sequence (Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951). The primary structural constraints have been determined in the P-loop located in the beta-subunit of the mitochondrial ATPase from yeast. The primary structural constraints were determined for 9 residues that form the P-loop, 190Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr-Val198. Each residue was tested individually for possible functional replacements while keeping the primary structure of the remainder of the molecule constant. This analysis indicates with greater than 95% confidence that Gly190,Gly195, and Lys196 are invariant and Thr197 can only be replaced with Ser. The most alterable residue is Gly191, where 10 replacements, even Phe, form a functional enzyme. The remaining positions allow some amino acid replacements while restricting others. The primary structural constraints of the P-loop of the mitochondrial F1 suggests that the three-dimensional structure of the P-loop is similar to that of ras.
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PMID:Primary structural constraints of P-loop of mitochondrial F1-ATPase from yeast. 814 26

Certain forms of ceroid lipofuscinosis, a hereditary degenerative disease, are characterized by accumulation of large amounts of subunit c of mitochondrial ATP synthase in lysosomal storage bodies of numerous tissues. The subunit c protein appears to constitute a major fraction of the total storage body protein. In previous studies it was demonstrated that hydrolysates of total storage body protein from affected humans and sheep contain significant amounts of epsilon-N-trimethyllysine (TML). This finding suggested that one or both of the two lysine residues of subunit c might be methylated in the stored form of the protein. The normal subunit c protein from mitochondria does not appear to be methylated. Using a putative canine model for the juvenile form of ceroid lipofuscinosis, analyses were conducted to determine whether lysosomal storage of subunit c was accompanied by lysine methylation of this protein. In affected dogs, as in humans and sheep with hereditary ceroid lipofuscinosis, the storage bodies were found to contain large amounts of subunit c protein, as indicated by polyacrylamide gel electrophoresis and partial amino acid sequence analysis. The subunit c protein partially purified from isolated storage bodies was found to contain lysine and TML in an almost equimolar ratio. Normal subunit c contains 2 lysine residues, one at position 7 and the other at position 43. Removal of the first 7 residues of the partially purified protein through sequential Edman degradation resulted in a dramatic increase in the TML to lysine ratio in the residual protein. This suggests that lysine residue 43 is methylated. Confirmation that residue 43 of the stored protein is TML was obtained by amino acid sequence analysis after cleavage of the protein with trypsin. This finding strongly suggests that specific methylation of lysine residue 43 of mitochondrial ATP synthase plays a central role in the lysosomal storage of this protein.
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PMID:Lysine methylation of mitochondrial ATP synthase subunit c stored in tissues of dogs with hereditary ceroid lipofuscinosis. 814 84

Vanadyl, (V = O)2+, is able to substitute for Mg2+ as a cofactor for ATPase activity catalyzed by the chloroplast F1-ATPase (CF1). Mg2+-dependent ATPase activity was also observed with CF1 that contained VO(2+)-ATP bound specifically to the noncatalytic N2 site. Modulation of the Mg(2+)-ATPase activity induced by VO2+ bound at this site indicates that the metal bound to the noncatalytic site affects catalytic activity. When CF1 is depleted of nucleotides from all but the N1 site, a single Mg2+ remains bound at a site designated M1. Addition of VO2+ to the depleted protein gives rise to an EPR spectrum characteristic of a CF1-bound VO2+ species. The binding curve of the VO2+ complex to latent, nucleotide-depleted CF1 was determined by the integrated intensities of the -5/2 parallel peak in the EPR spectrum as calibrated using atomic absorption spectroscopy. Under these conditions, VO2+ binds cooperatively to approximately two sites designated M2 and M3. Three-pulse ESEEM spectra of the CF1-VO2+ complex contain two intense modulations with frequencies and field-dependent behavior that show that they are from a directly coordinated 14N nucleus. Analysis of the bound VO2+ by ENDOR spectroscopy revealed the presence of a single group of protons associated with an equatorial amino or water ligand that is exchangeable with solvent. Using the additivity relation for hyperfine coupling, the most probable set of equatorial ligands to the VO2+ bound to CF1 under these conditions consists of one lysine nitrogen, two carboxyl oxygens from aspartate or glutamate, and one water.
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PMID:Characterization of ligands of a high-affinity metal-binding site in the latent chloroplast F1-ATPase by EPR spectroscopy of bound VO2+. 816 51


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