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)

When beef heart mitochondrial F1-ATPase is photoirradiated in the presence of 2-azido[alpha-32P]adenosine diphosphate, the beta subunit of the enzyme is preferentially photolabeled [Dalbon, P., Boulay, F., & Vignais, P. V. (1985) FEBS Lett. 180, 212-218]. The site of photolabeling of the beta subunit has been explored. After cyanogen bromide cleavage of the photolabeled beta subunit, only the peptide fragment extending from Gln-293 to Met-358 was found to be labeled. This peptide was isolated and digested by trypsin or Staphylococcus aureus V8 protease. Digestion by trypsin yielded four peptides, one of which spanned residues Ala-338-Arg-356 and contained all the bound radioactivity. When trypsin was replaced by V8 protease, a single peptide spanning residues Leu-342-Met-358 was labeled. Edman degradation of the two labeled peptides showed that radioactivity was localized on the following four amino acids: Leu-342, Ile-344, Tyr-345, and Pro-346.
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PMID:Identification of amino acid residues photolabeled with 2-azido[alpha-32P]adenosine diphosphate in the beta subunit of beef heart mitochondrial F1-ATPase. 287 32

A mutant strain KF87 of E. coli with a defective beta-subunit (Ala-151----Val) of F1-ATPase was isolated. The mutation is within the conserved sequence (G-X-X-X-X-G-K-T/S) of nucleotide-binding proteins. The mutant F1-ATPase had a much higher rate of uni-site hydrolysis of ATP than the wild type, and about 6% of the wild-type multi-site activity. The mutant enzyme showed defective transmission of conformational change(s) between the ligand- and aurovertin-binding sites.
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PMID:Beta-subunit of Escherichia coli F1-ATPase. An amino acid replacement within a conserved sequence (G-X-X-X-X-G-K-T/S) of nucleotide-binding proteins. 288 26

ATP synthase from bovine mitochondria is a complex of 13 different polypeptides, whereas the Escherichia coli enzyme is simpler and contains eight subunits only. Two of the bovine subunits, b and d, which had not been characterized, have been isolated from the purified enzyme. Subunits with sizes corresponding to bovine subunits b and d are evident in preparations of the enzyme from mitochondria of other species. Partial protein sequences have been determined by direct methods. On the basis of some of this information, two oligonucleotide mixtures, 17 and 18 bases in length, have been synthesized and used as hybridization probes in the isolation of clones of the cognate cDNAs. The sequences of the two proteins have been deduced from their DNA sequences. Subunit b is 214 amino acid residues in length and has a free N terminus. Subunit d is 160 amino acid residues long. Its N-terminal alanine is blocked by an N-acetyl group, as demonstrated by fast atom bombardment mass spectrometry of N-terminal peptides. The sequence near the N terminus of the b subunit is made predominantly of hydrophobic residues, whereas the remainder of the protein is mainly hydrophilic. This N-terminal hydrophobic region may be folded into an alpha-helical structure spanning the lipid bilayer. In its distribution of hydrophobic residues, this protein resembles the b subunits of ATP synthase complexes in bacteria and chloroplasts. The b subunit in E. coli forms an important structural link between the extramembrane sector of the enzyme F1, and the intrinsic membrane domain, FO. It is proposed that the bovine mitochondrial subunit b serves a similar function. If this is so, the mitochondrial enzyme, as the chloroplast ATP synthase, contains equivalent subunits to all eight of those that constitute the E. coli enzyme. Subunit d has no extensive hydrophobic sequences, and is not apparently related to any subunit described in the simpler ATP synthases in bacteria and chloroplasts.
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PMID:ATP synthase from bovine mitochondria. The characterization and sequence analysis of two membrane-associated sub-units and of the corresponding cDNAs. 289 Jul 67

The amino acid sequence -Gly-X-X-X-X-Gly-Lys- occurs in many, diverse, nucleotide-binding proteins, and there is evidence that it forms a flexible loop which interacts with one or other of the phosphate groups of bound nucleotide. This sequence occurs as -Gly-Gly-Ala-Gly-Val-Gly-Lys- in the beta-subunit of the enzyme F1-ATPase, where it is thought to form part of the catalytic nucleotide-binding domain. Mutants of Escherichia coli were generated in which residue beta-lysine 155, at the end of the above sequence, was replaced by glutamine or glutamate. Properties of the soluble purified F1-ATPase from each mutant were studied. The results showed: 1) replacement of lysine 155 by Gln or Glu decreased the steady-state rate of ATP hydrolysis by 80 and 66%, respectively. 2) Characteristics of ATP hydrolysis at a single site were not markedly changed in the mutant enzymes, implying that lysine 155 is not directly involved in bond cleavage during ATP hydrolysis or bond formation during ATP synthesis. 3) The binding affinity for MgATP was weakened considerably in the mutants (Lys much much greater than Gln greater than Glu), whereas the binding affinity for MgADP was affected only mildly (Lys = Gln greater than Glu), suggesting that lysine 155 interacts with the gamma-phosphate of ATP bound at a single high affinity catalytic site. 4) The major determinant of inhibition of steady-state ATPase turnover rate in the mutant enzymes was an attenuation of positive catalytic cooperativity. 5) The data are consistent with the idea that during multisite catalysis residue 155 of beta-subunit undergoes conformational movement which changes substrate and product binding affinities.
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PMID:Directed mutations of the strongly conserved lysine 155 in the catalytic nucleotide-binding domain of beta-subunit of F1-ATPase from Escherichia coli. 289 6

Three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli were produced by site-directed mutagenesis. These mutations directed the substitution of Glu-219 by Gln, or of Lys-203 by Ile, or of Glu-196 by Ala. Strains carrying either the Lys-203 or Glu-196 substitutions showed growth characteristics indistinguishable from the coupled control strain. Properties of membrane preparations from these strains were also similar to those from the coupled control strain. The substitution of Glu-219 by Gln resulted in a strain which was unable to utilise succinate as sole carbon source and had a growth-yield characteristic of an uncoupled strain. Membrane preparations of the Glu-219 mutant were proton impermeable and the F1-ATPase activity was inhibited by about 50% when membrane-bound. The results are discussed with reference to a previously proposed intramembranous proton pore involving subunits a and c.
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PMID:The proton pore in the Escherichia coli F0F1-ATPase: substitution of glutamate by glutamine at position 219 of the alpha-subunit prevents F0-mediated proton permeability. 289 67

The mitochondrial ATPase is rapidly inactivated by the arginine selective reagent phenylglyoxal. Recently, the purported major reacting residue has been reported for the chloroplast enzyme (Viale, A. M., and Vallejos, R. H. (1985) J. Biol. Chem. 260, 4958-4962) corresponding to Arg-328 in the beta-subunit of the yeast Saccharomyces cerevisiae mitochondrial ATPase, a highly conserved residue in the ATPase. This arginine residue was concluded to be in the active site of the ATPase and possibly involved in the binding of nucleotides. To test this hypothesis, site-directed mutagenesis of the yeast enzyme has been used to replace Arg-328 with alanine and lysine. The modified genes were transformed into a yeast strain, DMY111, which contained a null mutation in the gene coding for the beta-subunit of the ATPase. Both of the substitutions were functional in vivo as demonstrated by the ability of yeast transformants to grow on a nonfermentable carbon source. The water soluble F1-ATPase with Ala-328 and Lys-328 were extremely unstable, but could be stabilized with glycerol. The rate of enzymatic decay followed first order kinetics with half-lives of 1.1 and 4.0 min for the mutants with Ala-328 and Lys-328 in 10% and 5% glycerol, respectively, while the wild type enzyme was stable even in the absence of glycerol. Kinetic analysis of both ATPase and GTPase has been determined. The wild type enzyme had two observable apparent Km and Vmax values for ATPase which were 0.056 mM-1 and 67 units/min/mg and 0.140 mM-1 and 100 units/min/mg. The mutant enzyme containing Lys-328 showed similar kinetic values of 0.066 mM-1 and 23 units/min/mg and 0.300 mM-1 and 43 units/min/mg. The mutant enzyme containing Ala-328, however, only demonstrated a single site with values of 0.121 mM-1 and 45 units/min/mg. In contrast to ATPase activity, kinetic values for GTPase were nearly identical for the wild type and mutant enzymes. Opposite to predicted results, the mutant enzymes were more sensitive to the reagent phenylglyoxal. These results indicate that Arg-328 is important for protein stability, but not involved in catalysis.
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PMID:Arginine 328 of the beta-subunit of the mitochondrial ATPase in yeast is essential for protein stability. 289 71

Peptides generated from enzymatic hydrolysis of chicken enolase and the alpha- and beta-subunits of bovine F1-ATPase were analyzed by mass spectrometry to determine the nature of their modified N-termini. In the case of chicken enolase, a peptide was isolated from a Staphylococcus aureus proteinase digest by HPLC and analyzed directly by fast atom bombardment mass spectrometry (FABMS). In conjunction with mass spectral evidence obtained from the methyl ester derivative and a secondary tryptic peptide, a structure is proposed containing an N-acetyl serine at the N-terminus. The alpha-subunit of bovine mitochondrial ATPase was chromatographed by HPLC after S. aureus proteinase digestion and a single peak was analyzed on the basis of predicted retention times. A Mr 716 was determined by FABMS and pyrrolidone carboxylic acid was deduced on the basis of its amino acid composition and partial Edman sequence data. The beta-subunit of ATPase produced a series of closely eluting peaks on HPLC after limited digestion with trypsin of the alpha 2 beta 2 complex. These peptides were analyzed by both Edman degradation and FABMS. These data showed the N-terminus to be frayed with N-terminal sequences beginning in pyro-Glu-Ala-Ser, Gln-Ala-Ser, Glu-Ala-Ser, Ala-Ser, and Ser but with no N-acetyl-Ser as was previously thought.
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PMID:Structural elucidation of N-terminal post-translational modifications by mass spectrometry: application to chicken enolase and the alpha- and beta-subunits of bovine mitochondrial F1-ATPase. 289 18

Complementary DNA clones of prolidase (imidodipeptidase, EC 3.4.13.9) were isolated from human liver and placental cDNA libraries. Two clones named lambda PL21 and lambda PP6 from the liver and placental cDNA libraries, respectively, were analyzed in detail. The first clone, lambda PL21, carried a cDNA insert of 1.7 kilobase pairs and covered all the coding region of human prolidase mRNA. The second clone, lambda PP6, contained a 1.8-kilobase insert with a full-length 3'-untranslated region. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the two clones with the partial amino acid sequence determined by Edman degradation of peptides derived from human erythrocyte prolidase established that both clones code for human prolidase. The amino terminus of the human mature enzyme is blocked and seems to begin with the sequence X-Ala-Ala-Ala. Presumably no processing occurs at the carboxyl terminus. The mature enzyme is composed of 492 residues, corresponding to Mr 54,305. The sequence of prolidase is unique and not similar to any known protein, except for a significant similarity to regions of F1-ATPase alpha and beta subunits from various sources. The gene has been mapped to the short arm of chromosome 19 (19p13.2). Elucidation of the complete amino acid sequence and the gene location of prolidase should provide the basis for understanding structure-function relationships and also inherited disorders caused by deficiency of this metabolically important enzyme.
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PMID:Primary structure and gene localization of human prolidase. 292 54

When the bovine mitochondrial F1-ATPase is inactivated with dicyclohexyl[14C]carbodiimide and then gel-filtered, from 2 to 3 g atoms of 14C are incorporated/mol of enzyme. Prior inactivation of the enzyme by the modification of an essential tyrosine residue with 4-chloro-7-nitrobenzofurazan, a reaction that can be reversed by thiols, does not affect the irreversible inactivation of the ATPase by dicyclohexyl[14C]carbodiimide. During the large scale modification of the F1-ATPase by dicyclohexyl[14C]carbodiimide which led to 70% inactivation, 1.9 g atoms of 14C were incorporated/mol of enzyme. Isolation of the alpha, beta, and gamma subunits from this large scale inactivation revealed that the gram atoms of 14C bound per mol of each of the subunits was: alpha, 0.04; beta, 0.56; and gamma, 0.04. The majority of the radioactivity in a cyanogen bromide digest of the 14C-labeled beta subunit was isolated in a fragment that has the following amino acid sequence: Glu-Leu-Ile-Asn-Asn-Val-Ala-Lys-Ala-His-Gly-Gly-Tyr-Ser-Val-Phe-Ala-Gly-Val-Gly -Glu-Arg-Thr-Arg-Glu-Gly-Asn-Asp-Leu-Tyr-Glu*-His-Met; where Glu* represents the N gamma-glutamyl derivative of dicyclohexyl[14C]urea.
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PMID:Inactivation of the bovine mitochondrial F1-ATPase with dicyclohexyl[14C]carbodiimide leads to the modification of a specific glutamic acid residue in the beta subunit. 611 57

The uncE410 allele differs from the normal uncE gene in that C leads to T base changes occur at nucleotides 190 and 191, resulting in proline at position 64 in the c-subunit of the F1F0-ATPase being replaced by leucine. Two partial-revertant strains were isolated in which alanine-20 of the c-subunit was replaced by proline, owing to a G leads to C base change at nucleotide 58. These c-subunits, coded for by the uncE501 and uncE502 alleles, therefore contained two amino acid changes, namely proline-64 leads to leucine, and alanine-20 leads to proline. Membranes prepared from the partial-revertant strains lacked ATP-dependent atebrin-fluorescence-quenching activity but were able to carry out oxidative phosphorylation. The ATPase activity of the F1-ATPase was inhibited when bound to membranes from strains carrying the uncE410, uncE501 and uncE502 alleles. It is concluded that a bend in the helix axis in one of the arms of the c-subunit hairpin structure is required for integration of the c-subunit into a functional F1F0-ATPase.
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PMID:The F1F0-ATPase of Escherichia coli. Substitution of proline by leucine at position 64 in the c-subunit causes loss of oxidative phosphorylation. 619 78


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