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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

F1-ATPase is the major enzyme for ATP synthesis in mitochondria, chloroplasts, and bacterial plasma membranes. F1-ATPase obtained from thermophilic bacterium PS3 (TF1) is the only ATPase which can be reconstituted from its primary structure. Its beta subunit constitutes the catalytic site, and is capable of forming hybrid F1's with E. coli alpha and gamma subunits. Since the stability of TF1 resides in its primary structure, we cloned a gene coding for TF1, and the primary structure of the beta subunit was deduced from the nucleotide sequence of the gene to compare the sequence with those of beta's of three major categories of F1's; prokaryotic membranes, chloroplasts, and mitochondria. The following results were obtained. Homology: The primary structure of the TF1 beta subunit (473 residues, Mr = 51,995.6) showed 89.3% homology with 270 residues which are identical in the beta subunits from human mitochondria, spinach chloroplasts, and E. coli. It contained regions homologous to several nucleotide-binding proteins. Secondary structure: The deduced alpha-helical (30.1%) and beta-sheet (22.3%) contents were consistent with those determined from the circular dichroism spectra. Residues forming reverse turns (Gly and Pro) were highly conserved among the F1 beta subunits. Substituted residues and stability of TF1: We compared the amino acid sequence of the TF1 beta subunit with those of the other F1 beta subunits mentioned above. The observed substitutions in the thermophilic subunit increased its propensities to form secondary structures, and its external polarity to form tertiary structure. Codon usage: The codon usage of the TF1 beta gene was found to be unique. The changes in codons that achieved these amino acid substitutions were much larger than those caused by minimal mutations, and the third letters of the optimal codons were either guanine or cytosine, except in codons for Gln, Lys, and Glu.
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PMID:Stable structure of thermophilic proton ATPase beta subunit. 288 Aug 41

Six mutant uncD alleles, affecting essential residues of the beta-subunit of Escherichia coli proton-ATPase, have been identified by intragenic complementation mapping, cloning, and DNA sequencing. Five of the mutations impair catalysis but do not cause structural perturbation of F1-ATPase. The amino acid substitutions found were as follows: uncD412, Gly-142----Ser; uncD430 and uncD431, both Arg-246----Cys; uncD478, Ser-174----Phe; and uncD484, Met-209----Ile. Kinetic characteristics of each corresponding mutant F1-ATPase are described or reviewed. In each case, the major determinant of impaired catalysis appears to be an attenuation of positive catalytic site cooperativity. Additionally, each mutation affects intrinsic properties of the catalytic site, including affinity for ATP, the ratio between unisite-bound substrate and products, and the rate of release of product inorganic phosphate under unisite ATP hydrolysis conditions. These effects are discussed in terms of a structural model of the catalytic nucleotide-binding domain of beta-subunit proposed recently (Duncan, T.M., Parsonage, D., and Senior, A.E. (1986) FEBS Lett. 208, 1-6). Each of the mutations lies within that domain. The uncD409 allele abolishes normal assembly of F1-ATPase. The amino acid substitution is Gly-214----Arg, which is suggested to affect a beta-turn connecting a beta-strand and an alpha-helix in the predicted nucleotide-binding domain of the beta-subunit.
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PMID:The defective proton-ATPase of uncD mutants of Escherichia coli. Identification by DNA sequencing of residues in the beta-subunit which are essential for catalysis or normal assembly. 288 84

Oligonucleotide-directed mutagenesis was used to generate six mutant strains of Escherichia coli which had the following specific amino acid substitutions in the beta-subunit of F1-ATPase: (i) Lys-155----Gln; (ii) Lys-155----Glu; (iii) Gly-149----Ile; (iv) Gly-154----Ile; (v) Tyr-297----Phe;(vi) Tyr-354----Phe. The effects of each mutation on growth of cells on succinate plates or limiting (3 mM) glucose and on cell membrane ATPase activity and ATP-driven pH gradient formation were studied. The results showed Lys-155 to be essential for catalysis, as has been predicted previously from sequence homology and structural considerations; however, the results appear to contradict the hypothesis that Lys-155 interacts with one of the substrate phosphate groups because the Lys-155----Glu mutation was less detrimental than Lys-155----Gln. Gly-149 and Gly-154 have been predicted to be involved in essential conformational changes in F1-ATPase by virtue of their position in a putative glycine-rich flexible loop structure. The mutation of Gly-154----Ile caused strong impairment of catalysis, but the Gly-149----Ile mutation produced only moderate impairment. The two tyrosine residues chosen for mutation were residues which have previously received much attention due to their being the sites of reaction of the inactivating chemical modification reagents 4-chloro-7-nitrobenzofurazan (Tyr-297) and p-fluorosulfonylbenzoyl-5'-adenosine (Tyr-354). We found that mutation of Tyr-297----Phe caused only minor impairment of catalysis, and mutation of Tyr-354----Phe produced no impairment. Therefore, a direct role for either of these tyrosine residues in catalysis is unlikely.
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PMID:Directed mutagenesis of the beta-subunit of F1-ATPase from Escherichia coli. 288 16

A group of mutant uncA alleles, affecting essential residues of the alpha-subunit of Escherichia coli proton-ATPase, have been identified by intragenic complementation mapping, cloning, and DNA sequencing. One of the mutations, uncA450, abolishes normal assembly of F1-ATPase. The amino acid substitution found was Glu-299----Lys, which is predicted to lie in an alpha-helix in alpha-subunit. The reversal of the charge at residue 299 is a likely cause of defective assembly. The uncA462 allele causes impairment of catalysis while allowing normal assembly of membrane-bound F1-ATPase. The amino acid substitution found was Ser-347----Phe. Three mutations which impair catalysis but do not cause structural perturbation of either membrane-bound or solubilized F1ATPase were characterized as follows: uncA401, Ser-373----Phe; uncA447, Gly-351----Asp; uncA453, Ser-375----Phe. We predict here that the nucleotide-binding domain of alpha-subunit is formed by the amino acids in the sequence from residue 160 to approximately residue 340. The mutations which cause impairment of catalysis lie in a short segment between residues 347-375 of alpha-subunit, at the C-terminal end of the predicted nucleotide-binding domain. This segment is suggested to be important for beta-alpha-beta intersubunit conformational interaction involved in positive catalytic cooperativity in F1-ATPase.
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PMID:The defective proton-ATPase of uncA mutants of Escherichia coli. Identification by DNA sequencing of residues in the alpha-subunit which are essential for catalysis or normal assembly. 288 25

Photolabeling of nucleotide binding sites in nucleotide-depleted mitochondrial F1 has been explored with 2-azido [alpha-32P]adenosine diphosphate (2-N3[alpha-32P] ADP). Control experiments carried out in the absence of photoirradiation in a Mg2+-supplemented medium indicated the presence of one high affinity binding site and five lower affinity binding sites per F1. Similar titration curves were obtained with [3H]ADP and the photoprobe 3'-arylazido-[3H]butyryl ADP [( 3H]NAP4-ADP). Photolabeling of nucleotide-depleted F1 with 2-N3[alpha-32P]ADP resulted in ATPase inactivation, half inactivation corresponding to 0.6-0.7 mol of photoprobe covalently bound per mol F1. Only the beta subunit was photolabeled, even under conditions of high loading with 2-N3[alpha-32P]ADP. The identification of the sequences labeled with the photoprobe was achieved by chemical cleavage with cyanogen bromide and enzymatic cleavage by trypsin. Under conditions of low loading with 2-N3[alpha-32P]ADP, resulting in photolabeling of only one vacant site in F1, covalently bound radioactivity was located in a peptide fragment of the beta subunit spanning Pro-320-Met-358 identical to the fragment photolabeled in native F1 (Garin, J., Boulay, F., Issartel, J.-P., Lunardi, J., and Vignais, P. V. (1986) Biochemistry 25, 4431-4437). With a heavier load of photoprobe, leading to nearly 4 mol of photoprobe covalently bound per mol F1, an additional region of the beta subunit was specifically labeled, corresponding to a sequence extending from Gly-72 to Arg-83. The isolated beta subunit also displayed two binding sites for 2-N3-[alpha-32P]ADP. When F1 was first photolabeled with a low concentration of NAP4-ADP, leading to the covalent binding of 1.5 mol of NAP4-ADP/mol F1, with the bound NAP4-ADP distributed equally between the alpha and beta subunits, a subsequent photoirradiation in the presence of 2-N3[alpha-32P]ADP resulted in covalent binding of the 2-N3[alpha-32P]ADP to both alpha and beta subunits. It is concluded that each beta subunit in mitochondrial F1 contains two nucleotide binding regions, one of which belongs to the beta subunit per se, and the other to a subsite shared with a subsite located on a juxtaposed alpha subunit. Depending on the experimental conditions, the subsite located on the alpha subunit is either accessible or masked. Unmasking of the subsite in the three alpha subunits of mitochondrial F1 appears to proceed by a concerted mechanism.
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PMID:Mapping of nucleotide-depleted mitochondrial F1-ATPase with 2-azido-[alpha-32P]adenosine diphosphate. Evidence for two nucleotide binding sites in the beta subunit. 288 35

The mutation Gly-29----Asp in the alpha-subunit of the F1-ATPase from Escherichia coli was characterized and shown to cause the following effects. 1) Oxidative phosphorylation was markedly impaired in vivo 2) Membrane ATPase and ATP-driven proton-pumping activities were decreased markedly. 3) Membranes were proton-permeable, and membrane-bound ATPase was dicyclohexylcarbodiimide-insensitive. Therefore, it appeared that integration between F1 and F0 was abnormal. This was confirmed directly by the demonstration that the mutant F1 bound poorly to stripped membranes from a normal strain. Purified, soluble mutant F1 had normal ATPase activity. These results suggest that residue Gly-29, which is strongly conserved in alpha-subunits of F1-ATPases, lies in a region of the alpha-subunit important for membrane binding. Thus, three regions of the F1-alpha-subunit have now been recognized, specialized for membrane binding, nucleotide binding, and alpha/beta intersubunit signal transmission, respectively. The approximate locations of the three regions are described.
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PMID:A mutation in the alpha-subunit of F1-ATPase from Escherichia coli affects the binding of F1 to the membrane. 289 4

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

Residues beta Glu-181 and beta Glu-192 of E. coli F1-ATPase (the DCCD-reactive residues) were mutated to Gln. Purified beta Gln-181 F1 showed 7-fold impairment of 'unisite' Pi formation from ATP and a large decrease in affinity for ATP. Thus the beta-181 carboxyl group in normal F1 significantly contributes to catalytic site properties. Also, positive catalytic site cooperativity was attenuated from 5 X 10(4)- to 548-fold in beta Gln-181 F1. In contrast, purified beta Gln-192 F1 showed only 6-fold reduction in 'multisite' ATPase activity. Residues beta Gly-149 and beta Gly-154 were mutated to Ile singly and in combination. These mutations, affecting residues which are strongly conserved in nucleotide-binding proteins, were chosen to hinder conformational motion in a putative 'flexible loop' in beta-subunit. Impairment of purified F1-ATPase ranged from 5 to 61%, with the double mutant F1 less impaired than either single mutant. F1 preparations containing beta Ile-154 showed 2-fold activation after release from membranes, suggesting association with F0 restrained turnover on F1 in these mutants.
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PMID:E. coli F1-ATPase: site-directed mutagenesis of the beta-subunit. 289 2

Ca2+-Transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum contains several SH groups which are reactive with N-ethylmaleimide (MalNEt) at pH 7.0. The location of the one which is most reactive with MalNEt (SHN, Kawakita et al. J. Biochem. 87, 609 (1980)) was identified on the amino acid sequence of the ATPase. SHN was labeled by reacting sarcoplasmic reticulum membranes with [14C] MalNEt to a labeling density of 1 mol/mol ATPase. [14C]MalNEt-labeled membranes were digested with thermolysin and 14C-labeled SHN peptides were fractionated by Sephadex LH-20 chromatography to give two major peaks of radioactivity. [14C]-MalNEt-labeled peptides were further purified to homogeneity by C18-reversed phase HPLC. Two radioactive peptides containing modified cysteine (Cys), Leu-Gly-Cys-Thr-Ser and Val-Cys-Lys-Met, were finally obtained in roughly equal amounts and in reasonable recovery. Both of these sequences were found in the amino acid sequence of Ca2+-transporting ATPase (Brandl et al. Cell 44, 597 (1986)), and Cys344 and Cys364 were identified as the targets of MalNEt-modification. Thus, 0.5 mol/mol ATPase of each Cys residue actually reacted rapidly with MalNEt under the conditions leading to SHN-modification. Modification of either one with MalNEt may negatively affect the reactivity of the other. Both of the highly reactive SH groups are located in the neighborhood of Asp351, the phosphorylation site of ATPase.
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PMID:Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. I. Location of a group which is most reactive with N-ethylmaleimide. 295 11

We have developed a defined in vitro system for packaging phage T3 DNA which is composed of purified proheads and the noncapsid proteins gp18 and gp19, products of genes 18 and 19 (K. Hamada, H. Fujisawa, and T. Minagawa, 1986, Virology 151, 119-123). The in vitro system displayed an ATPase activity. The requirements for ATPase activity were the same as those for DNA packaging. ATPase was inhibited by a nonhydrolyzable ATP analog, adenosine-5'-O-(3'-thiotriphosphate) (ATP-gamma-S). ATPase activity did not display specificity for T3 DNA. A reaction mixture containing 8- proheads, proheads deficient in gp8, a portal protein for DNA entrance, or mature heads had no gp18- gp19-dependent ATPase activity. gp8 itself had no ATPase activity and did not complement 8- proheads for ATPase activity. Photoaffinity labeling of proheads, gp18 and gp19 with 8-azidoadenosine-5'-[alpha-32P]triphosphate([32P]8-N3ATP) resulted in preferential labeling of gp19. Protection from incorporation of [32P]8-N3ATP was afforded by ATP but not by AMP and ADP. From these results, it is concluded that gp19 has an ATP binding site(s). A conserved sequence of ATP-binding site containing Gly-X-Gly-X-X-Gly-X-Val is found in gp19.
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PMID:Characterization of ATPase activity of a defined in vitro system for packaging of bacteriophage T3 DNA. 295 57


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