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)

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

The original aim of the review has been to probe into the validity of the paradigm on the high energy-carrier function of ATP. It seemed to be called into question on the basis of findings with H(+)-transporting ATP synthase suggesting the formation of ATP from ADP and Pi without energy input. Thus, ATP appeared as a low-energy compound. Starting from the current, rich knowledge of the molecular structure and the inviting thinking on the mechanism of H(+)-transporting ATP synthase, we have endeavoured to freshly interpret and integrate the pertinent observations in the light of the comprehensively derived model of the molecular mechanism of energy interconversion by Na+/K(+)-transporting ATPase. In this way, we have uncovered the common mechanistic elements of the two energy-interconverting enzymes. The emerging purpose of the present paper has been the 'synthesis' of a self-contained concept of the molecular mechanism of the interconversion of electrochemical and chemical Gibbs energies by H(+)-transporting ATP synthase. The outcome is reflected in the following tentative evaluations. 1. In ATP hydrolysis, the great Gibbs energy change which is observed in solution, is largely conserved by the F1 sector of ATP synthase as mechanical Gibbs energy in the enzyme's protein fabric, so that it can be utilized in the resynthesis of ATP from enzyme-bound ADP and Pi. The plainly measured low Gibbs energy change results from large compensating enthalpy and entropy changes that reflect the underlying changes in protein conformation. 2. In stoichiometric ATP synthesis by F1 sector from ADP and Pi bound to the catalytic centre, their intrinsic binding energy brings about a loss of peptide chain entropy that makes possible an entropy-driven ATP formation. 3. The driving force for ATP synthesis cannot be the high Gibbs energy change on binding of product ATP; the tight ATP-enzyme complex rather is a low Gibbs energy intermediate from which escape is difficult. 4. The catalytic centre exists either in an open state unable to firmly bind the substrate-product couple, or in a closed state protecting formed ATP from facile hydrolysis by ambient water. 5. The cleft closure, induced by binding of Pi and ADP or ATP, does not necessarily need external energy supply, because the cleft closure proceeds from rigid domain rotations which can occur rather spontaneously. In further analogy to adenylate kinase, the driving force of this domain movement presumably comes from the electrostatic interactions between phosphate moieties and arginine side chains in the catalytic centre.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Synthesis of a self-contained concept of the molecular mechanism of energy interconversion by H(+)-transporting ATP synthase. 805 42

We introduced mutations to test the function of the hydrophobic sector of subunit 4 from Saccharomyces cerevisiae ATP synthase. Mutations were introduced at the chromosomic locus by homologous transformation of a strain disrupted in the ATP4 gene. The strain carrying the replacement Leu68-Val69-->Arg-Glu did not grow at 37 degrees C owing to a lack of assembly of F1 and Fo sectors at this temperature. The mutant strain grew slowly by oxidative phosphorylation at 28 degrees C with a growth yield 30% lower than the wild type. Analysis of the mutant strain showed a homogeneous population of altered ATP synthase with an energy coupling impairment. The mutant strain was oligomycin-resistant since the I50 value of oligomycin inhibition of ATPase and ATP synthase activities was 2-3-fold higher than that of the wild type, thus showing an alteration of the target to oligomycin. The level of phosphorylation or ATP induced a proton-dissipating pathway through Fo, which was insensitive to oligomycin but was sensitive to dicyclohexylcarbodiimide, thus suggesting an alteration in the regulation of ATP synthase proton permeability by the catalytic sector. From these results, we propose that the dicyclohexylcarbodiimide inhibition site is located upstream of the oligomycin inhibition site when considering the proton flux occurring during ATP synthesis.
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PMID:Mutation in the hydrophobic domain of ATP synthase subunit 4 (subunit b) of yeast mitochondria disturbs coupling between proton translocation and catalysis. 806 46

The sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser) is conserved in nucleotide binding proteins including the alpha and beta subunits of the ATP synthase. Various mutations were introduced in the alpha Lys-175 and alpha Thr-176 residues in the sequence (Gly-Asp-Arg-Gln-Thr-Gly-Lys-Thr, residues 169-176) of the Escherichia coli ATP synthase alpha subunit. Surprisingly, single amino acid substitutions drastically affected the subunit assembly of the enzyme. The entire enzyme assembly was lost by alpha Lys-175-->Phe (or Trp) or alpha Thr-176-->Phe (or Tyr) mutation. Other mutants had similar (alpha His-175, alpha Ser-175, alpha Gly-175, alpha Ser-176, and alpha His-176 mutants) or lower (alpha Ala-176, alpha Cys-176, alpha Leu-176, and alpha Val-176 mutants) effects on assembly of the active enzyme compared with that of the wild-type. However, all these mutant enzymes except the alpha Ser-176 enzyme showed enhanced cold sensitivities and reduced stabilities at high temperature. Mutant enzymes such as alpha Gly-175 and alpha His-176 showed low multi-site (steady state) catalysis, possibly due to loss of proper subunit-subunit interactions. These results suggest that the alpha Lys-175 and alpha Thr-176 residues are not absolutely essential for catalysis, but that they, or possibly the entire conserved sequence, are located in the key domain for the subunit-subunit interactions essential for enzyme stability and steady state activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The alpha subunit of ATP synthase (F0F1): the Lys-175 and Thr-176 residues in the conserved sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser) are located in the domain required for stable subunit-subunit interaction. 826 95

The human and bovine genomes each contain two expressed nuclear genes, called P1 and P2, for subunit c, a hydrophobic subunit of the membrane sector, Fo, of mitochondrial ATP synthase. Both P1 and P2 encode the same mature protein, but the associated mitochondrial import sequences are different. In sheep with the neurodegenerative disease ceroid lipofuscinosis, and also in humans with Batten's disease, unmodified subunit c accumulates in lysosome-derived organelles in a variety of tissues. However, the sequences of cDNAs for P1 and P2 from sheep with ceroid lipofuscinosis were identical to those in healthy control animals. Therefore, since there was no mutation in either of the mitochondrial import sequences of subunit c in the diseased animals, ceroid lipofuscinosis does not arise from changes in an import sequence causing mis-targeting of the c subunit to lysosomes. The levels of expression of P1 and P2 genes were approximately the same in diseased and healthy animals, and so the protein is unlikely to accumulate because of excessive transcription of either gene. Transcription of a spliced pseudogene related to P2 was detected in both a control animal and a sheep with ceroid lipofuscinosis. The transcripts encode amino acids 1-31 of the P2 mitochondrial targeting sequence. In the diseased animal, an arginine replaced a glutamine in the control sequence. However, restriction fragment analysis of genomic DNA from a further 12 sheep established that the sequence differences were not linked to ceroid lipofuscinosis.
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PMID:Characterization of the expressed genes for subunit c of mitochondrial ATP synthase in sheep with ceroid lipofuscinosis. 832 73

Site-directed mutagenesis was used to investigate the roles of three proline residues (Pro-103, Pro-122 and Pro-143) in the a-subunit of the E. coli F0F1-ATPase. All three were found to have a role in stabilizing the a-subunit structure in that removal of the F1-ATPase from membranes prepared from each of the mutant strains resulted in the loss of passive proton translocation activity. Pro-103 is predicted to be within a transmembrane helix. Pro-122 and Pro-143 are located just outside the membrane and near two residues (Asp-124 and Arg-140) previously proposed to form a charge pair. The phenotype of mutants in which Pro-122 or Pro-143 were replaced by alanine was similar to previously isolated mutants affected in Asp-124 and Arg-140. This suggested that the main effect of the mutations was to destroy the charge pair between Asp-124 and Arg-140. Double mutants resulting from all possible combinations of these four mutations were constructed and, with the exception of P122A + D124A, had a similar phenotype to the single mutants. This is consistent with the idea that all four single changes had the same effect on a-subunit structure. In contrast, combining the P122A or P143A changes with another mutation which caused a similar phenotype (D44N) resulted in a complete loss of oxidative phosphorylation.
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PMID:Functional stability of the a-subunit of the F0F1-ATPase from Escherichia coli is affected by mutations in three proline residues. 834 58

Each of three conserved positively-charged residues in the C-terminal region of subunit 8 of yeast (Saccharomyces cerevisiae) mitochondrial ATP synthase was replaced with isoleucine. The assembly and functional properties of the resulting variants (substituted at Arg-37, Arg-42 and Lys-47) were examined using in-vitro systems to assay import into isolated mitochondria and to monitor assembly into ATP synthase, as well as an in-vivo rescue system using host yeast cells lacking endogenous subunit 8. Each such variant was found to be impaired in assembly in vitro, after import in the form of a chimaeric protein bearing a leader sequence with mitochondrial targeting function. Import precursors bearing a duplicated-leader sequence, engendering enhanced delivery to mitochondria of the passenger variant subunit-8 proteins, enabled assembly of the (Lys-47-->Ile) variant to be detected in vitro but not that of (Arg-37-->Ile) or (Arg-42-->Ile) variants. The respiratory growth of subunit 8-deficient host cells could be rescued with the (Lys-47-->Ile) variant expressed allotopically in the nucleus. Such rescued cells were found to have an enhanced growth rate (comparable to that produced by non-mutagenized parental subunit 8) when delivered to mitochondria with the duplicated-leader sequence, as compared to the single-leader sequence. This confirms that the impediment in the (Lys-47-->Ile) variant lies in the efficiency of its assembly, rather than a functional defect, as such, arising from the loss of that positive charge. In contrast, host cells were unable to be rescued by the (Arg-37-->Ile) and (Arg-42-->Ile) variants, even when they were endowed with the duplicated leader sequence. It is concluded that the positively-charged C-terminal domain of subunit 8, common to fungal and mammalian homologues of this protein, plays a key role in its assembly into mitochondrial ATP synthase.
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PMID:Each of three positively-charged amino acids in the C-terminal region of yeast mitochondrial ATP synthase subunit 8 is required for assembly. 834 59

The a subunit of F1F0 ATP synthase contains a highly conserved region near its carboxyl terminus which is thought to be important in proton translocation. Cassette site-directed mutagenesis was used to study the roles of four conserved amino acids Gln-252, Phe-256, Leu-259, and Tyr-263. Substitution of basic amino acids at each of these four sites resulted in marked decreases in enzyme function. Cells carrying a subunit mutations Gln-252-->Lys, Phe-256-->Arg, Leu-259-->Arg, and Tyr-263-->Arg all displayed growth characteristics suggesting substantial loss of ATP synthase function. Studies of both ATP-driven proton pumping and proton permeability of stripped membranes indicated that proton translocation through F0 was affected by the mutations. Other mutations, such as the Phe-256-->Asp mutation, also resulted in reduced enzyme activity. However, more conservative amino acid substitutions generated at these same four positions produced minimal losses of F1F0 ATP synthase. The effects of mutations and, hence, the relative importance of the amino acids for enzyme function appeared to decrease with proximity to the carboxyl terminus of the a subunit. The data are most consistent with the hypothesis that the region between Gln-252 and Tyr-263 of the a subunit has an important structural role in F1F0 ATP synthase.
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PMID:Mutagenic analysis of the a subunit of the F1F0 ATP synthase in Escherichia coli: Gln-252 through Tyr-263. 838 11

The gamma subunit mutations, gamma Met-23-->Lys or Arg, in the Escherichia coli ATP synthase were previously reported to cause dramatically inefficient energy coupling between ATPase catalysis and H+ translocation (Shin, K., Nakamoto, R.K., Maeda, M., and Futai, M. (1992) J. Biol. Chem. 267, 20835-20839). In this paper, we report that second-site mutations in the gamma subunit can suppress the effects of gamma Met-23-->Lys. By screening randomly mutagenized uncG (gamma Met-23-->Lys), eight mutations in the carboxyl-terminal region were identified; strains carrying gamma Arg-242-->Cys, gamma Gln-269-->Arg, gamma Ala-270-->Val, gamma Ile-272-->Thr, gamma Thr-273-->Ser, gamma Glu-278-->Gly, gamma Ile-279-->Thr, or gamma Val-280-->Ala in combination with gamma Met-23-->Lys were able to grow by oxidative phosphorylation. H+ pumping assayed in membranes prepared from double mutation strains demonstrated that efficient ATP-dependent H+ transport was restored. Interestingly, the single mutations, gamma Gln-269-->Arg or gamma Thr-273-->Ser, caused reduced growth by oxidative phosphorylation; however, when these mutations were in combination with gamma Met-23-->Lys, growth was substantially increased. Furthermore, strains carrying gamma Met-23-->Lys, gamma Gln-269-->Arg, or gamma Thr-273-->Ser as single mutations were temperature sensitive, whereas, strains with the double mutations, gamma Met-23-->Lys/gamma Gln-269-->Arg or gamma Met-23-->Lys/gamma Thr-273-->Ser, were thermally stable. Taken together, these results strongly suggest that gamma Met-23, gamma Arg-242, and the region between gamma Gln-269 to gamma Val-280 are close to each other and interact to mediate efficient energy coupling.
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PMID:The gamma subunit of the Escherichia coli ATP synthase. Mutations in the carboxyl-terminal region restore energy coupling to the amino-terminal mutant gamma Met-23-->Lys. 841 64


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