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)

Mammalian cells differ considerably in the duration of anoxia which they can tolerate despite the fact that dramatic bioenergetic changes occur rapidly. Previous studies indicate that the ability to tolerate anoxia is at least partly due to an endogenous signal transduction system that senses O2 deficiency and signal altered ion transport functions in the mitochondria. The responses included inhibition of ATP synthase, ADP/ATP exchange, inorganic phosphate uptake, mitochondrial swelling, and loss of the mitochondrial proton-motive force. An important distinction between KCN toxicity and anoxia is that KCN does not elicit these protective mechanisms. Thus, the ability of a compound to elicit these mechanisms in KCN-treated cells provides an assay for potential agonists of the endogenous protective mechanisms.
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PMID:Protective effect of dicalciphor during mitochondrial failure. 150 61

1) Using a combination of site-directed mutagenesis and fluorescence spectroscopy we have studied the location and function of residue beta Y331 in the catalytic site of Escherichia coli F1-ATPase. The fluorescent analog lin-benzo-ADP was used as a catalytic-site probe, and was found to bind to three sites in normal F1, with Kd1 = 0.20 microM and Kd2,3 = 5.5 microM. lin-Benzo-ATP was a good substrate for hydrolysis. 2) The mutants investigated were beta Y331F, L, A and E. kcat/KM for ATP hydrolysis in purified F1 was reduced according to the series Y greater than or equal to F greater than L greater than A greater than E, with E being severely impaired; concomitant decreases in binding affinity for lin-benzo-ADP were seen. 3) Fluorescence properties of lin-benzo-ADP bound to F1 differed widely, depending on the residue present at position beta 331. Red shifts of excitation and emission spectra occurred with F and L residues, but not with Y, A, or E. There was strong quenching of fluorescence with wild-type (Y), partial quenching with A, and no quenching with F, L, or E. 4) We conclude that (a) the environment around the bound adenine moiety in the catalytic site is nonpolar, (b) residue beta 331 is part of the adenine-binding subdomain and when tyrosine is the residue, the phenolic hydroxyl makes direct interaction with the fluorophore, (c) an aromatic residue is not absolutely required at position beta 331 for catalytic function, but an increase in polarity leads to functional impairment, and (d) in terms of fluorescence response of bound lin-benzo-ADP all three catalytic sites behaved the same. 5) F1 from mutant beta Y297F bound lin-benzo-ADP with the same fluorescence and binding characteristics as normal F1, and catalytic properties were similar to normal. Therefore, there was no reason to conclude that residue beta Y297 is involved in binding the adenine moiety of ATP.
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PMID:On the location and function of tyrosine beta 331 in the catalytic site of Escherichia coli F1-ATPase. 153 Sep 42

Using manual rapid-mixing procedures in which small, equal volumes of Escherichia coli F1-ATPase and [gamma-32P]ATP were combined at final concentrations of 2 and 0.2 microM, respectively (i.e., unisite catalysis conditions), it was shown that greater than or equal to 66% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi equal to 0.4 and the rate of dissociation of bound [32P]Pi equal to 3.5 x 10(-3) s-1, similar to previously published values. Azide is known to inhibit cooperative but not unisite catalysis in F1-ATPase [Noumi, T., Maeda, M., & Futai, M. (1987) FEBS Lett. 213, 381-384]. In the presence of 1 mM sodium azide, 99% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi being 0.57. These experiments demonstrated that when conditions are used which minimize cooperative catalysis, most or all of the F1 molecules bind substoichiometric ATP tightly, hydrolyze it with retention of bound ATP and Pi, and release the products slowly. The data justify the validity of previously published rate constants for unisite catalysis. Unisite catalysis in E. coli F1-ATPase was studied at varied pH from 5.5 to 9.5 using buffers devoid of phosphate. Rate constants for ATP binding/release, ATP hydrolysis/resynthesis, Pi release, and ADP binding/release were measured; the Pi binding rate constant was inferred from the delta G for ATP hydrolysis. ATP binding was pH-independent; ATP release accelerated at higher pH. The highest KaATP (4.4 x 10(9) M-1) was seen at physiological pH 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Catalytic sites of Escherichia coli F1-ATPase. Characterization of unisite catalysis at varied pH. 153 Oct 27

(1) Dimethyl sulfoxide (DMSO) markedly inhibited the Vmax of multisite ATPase activity in Escherichia coli F1-ATPase at concentrations greater than 30% (v/v). Vmax/KM was reduced by 2 orders of magnitude in 40% (v/v) DMSO at pH 7.5, primarily due to reduction of Vmax. The inhibition was rapidly reversed on dilution into aqueous buffer. (2) KdATP at the first, high-affinity catalytic site was increased 1500-fold from 2.3 x 10(-10) to 3.4 x 10(-7) M in 40% DMSO at pH 7.5, whereas KdADP was increased 3.2-fold from 8.8 to 28 microM. This suggests that the high-affinity catalytic site presents a hydrophobic environment for ATP binding in native enzyme, that there is a significant difference between the conformation for ADP binding as opposed to ATP binding, and that the ADP-binding conformation is more hydrophilic. (3) Rate constants for hydrolysis and resynthesis of bound ATP in unisite catalysis were slowed approximately 10-fold by 40% DMSO; however, the equilibrium between bound Pi/bound ATP was little changed. The reduction in catalysis rates may well be related to the large increase in KdATP (less constrained site). (4) Significant Pi binding to E. coli F1 could not be detected either in 40% DMSO or in aqueous buffer using a centrifuge column procedure. (5) We infer, on the basis of the measured constants KaATP, K2 (hydrolysis/resynthesis of ATP), k+3 (Pi release), and KdADP and from estimates of k-3 (Pi binding) that delta G for ATP hydrolysis in 40% DMSO-containing pH 7.5 buffer is between -9.2 and -16.8 kJ/mol.
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PMID:Effects of dimethyl sulfoxide on catalysis in Escherichia coli F1-ATPase. 153 Oct 28

Energy-dependent activation of the chloroplast ATP synthase (CF0CF1) has been elucidated by investigating the conformational changes, the ADP effect, and the catalytic cooperativity of ATP hydrolysis. Conformational change was observed by measuring the reactivity of Lys-109 of the epsilon subunit of chloroplast coupling factor 1 with pyridoxal 5'-phosphate. In the postillumination dark, the Lys-109 reactivity decreased biphasically with half-times of less than 1 and 17 s. NH4Cl accelerated the slow phase decrease. Addition of ADP (0.2 microM) in the postillumination dark inactivated CF0CF1 (0.05 microM) with a half-time of 12 s. At high concentration of CF0CF1 (1.2 microM), inactivation occurred without exogenously added ADP with a half-time of 12 s. Accompanying the inactivation, the positive catalytic cooperativity of ATP hydrolysis decreased. Addition of 10 mM NH4Cl before ADP (0.2 microM) decelerated the ADP-induced inactivation to a half-time of 64 s. Throughout this inactivation, the positive catalytic cooperativity was maintained at a high level. These results suggest three distinct conformations of CF0CF1, EH, EM, and EL, and their ADP binding forms EM-ADP and EL-ADP. EH, EM, and EL have a low affinity for ADP, a high affinity for ADP, and low accessibility to ADP, respectively. EM and EL exhibit highly cooperative ATP hydrolysis. ATP hydrolysis catalyzed by EM-ADP exhibits no cooperativity. EL-ADP is inactive.
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PMID:Energy-dependent changes in conformation and catalytic activity of the chloroplast ATP synthase. 153 Nov 39

The interaction of 2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP) and TNP-ADP to F1-ATPase from a thermophilic bacterium PS3 (TF1) was investigated. When TNP-ADP or TNP-ATP was added to the isolated alpha or beta subunit of TF1, characteristic difference spectra were generated for each subunit. Difference spectra generated on addition of these analogs to TF1 resembled those observed for the beta subunit, indicating TNP analogs bind to the beta subunits in the molecule of TF1. Results of equilibrium dialysis showed that TNP-ADP binds to a single high affinity site on TF1 in the presence of Mg2+ with a dissociation constant of 2.2 nM. When TNP-ATP was added to TF1 in a substoichiometric molar ratio, it rapidly bound to TF1 and was slowly hydrolyzed. The hydrolysis proceeded nearly to completion without showing stable equilibrium between bound species of TNP-ATP and TNP-ADP. Similar to beef heart mitochondrial F1, this hydrolysis was greatly accelerated by the chase-addition of 100 microM ATP. However, the hydrolyzed product, TNP-ADP, remained bound on the beta subunit even after the chase.
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PMID:Single site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP by the F1-ATPase from thermophilic bacterium PS3 is accelerated by the chase-addition of excess ATP. 153 55

The F1-ATPase from Micrococcus lysodeikticus is isolated in the absence of exogenous nucleotides. After removing loosely bound nucleotides from the isolated enzyme by gel permeation chromatography, analysis for tightly bound nucleotides revealed in 14 experiments 0.4 +/- 0.1 mol ADP, 0.5 +/- 0.2 mol GDP, and 0.8 +/- 0.2 mol ATP per mol of F1. Incubation of the isolated enzyme with Mg2+ or Ca2+ did not alter the endogenous nucleotide composition of the enzyme, indicating that endogenous ATP is not bound to a catalytic site. Incubation of the enzyme with P(i) decreased the amount of tightly bound ADP and GDP but did not effect the ATP content. Hydrolysis of MgATP in the presence of sulfite raised the tightly bound ADP and lowered tightly bound GDP on the enzyme. In the reciprocal experiment, hydrolysis of MgGTP in the presence of sulfite raised tightly bound GDP and lowered tightly bound ADP. Turnover did not affect the content of tightly bound ATP on the enzyme. These results suggest that endogenous ADP and GDP are bound to exchangeable catalytic sites, whereas endogenous ATP is bound to noncatalytic sites which do not exchange. The presence of endogenous GDP on catalytic sites of isolated F1 suggests that the F0F1-ATP synthase of M. lysodeikticus might synthesize both GTP and ATP under physiological conditions. In support of this hypothesis, we have found that plasma membrane vesicles derived from M. lysodeikticus synthesize [32P]GTP from [32P]P(i) using malate as electron donor for oxidative phosphorylation.
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PMID:Significant quantities of endogenous GDP and ADP are present on catalytic sites of the F1-ATPase isolated from M. lysodeikticus in the absence of added nucleotides. 153 27

The single sulfhydryl residue (cysteine-63) of the beta subunit of the chloroplast ATP synthase F1 (CF1) was accessible to labeling reagents only after removal of the beta subunit from the enzyme complex. This suggests that cysteine-63 may be located at an interface between the beta and the alpha subunits of CF1, although alternative explanations such as a conformational change in beta brought about by its release from CF1 cannot be ruled out. Cysteine-63 was specifically labeled with [(diethylamino)methylcoumarinyl]-maleimide, and the distance between this site and trinitrophenyl-ADP at the nucleotide binding site on beta was mapped using fluorescence resonance energy transfer. Cysteine-63 is located in a hydrophobic pocket, 42 A away from the nucleotide binding site on beta.
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PMID:Structural mapping of cysteine-63 of the chloroplast ATP synthase beta subunit. 153 53

ADP-induced inhibition of mitochondrial F1-ATPase has been studied. It is shown that in the presence of magnesium and the absence of light, the photoaffinity ADP analog, 2-azido-ADP, induces a reversible inhibition of native F1 that is indistinguishable from that obtained with ADP. Photolysis of the inactive complex results in the predominant labeling of a catalytic-site peptide identified previously (Cross et al., 1987, Proc. Natl. Acad. Sci. USA 84, 5715-5719). Dissociation of the inactive complex formed between F1 and ADP is biphasic with a rapid azide-insensitive phase followed by a slow azide-sensitive phase (k approximately 3 x 10(-3) s-1). It is also shown that incubation of the ADP-inhibited enzyme with EDTA or phosphate does not result in release or migration of ADP from the catalytic site. However, it does convert the complex to a form that reactivates in the presence of 100 microM ATP at a rate too rapid to observe using manual mixing.
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PMID:Adenine nucleotide-binding sites on mitochondrial F1-ATPase: studies of the inactive complex formed upon binding ADP at a catalytic site. 153

(1) Previous mutational analyses have shown that residue beta R398 of the beta-subunit is a key residue for binding of the inhibitory antibiotic aurovertin to Escherichia coli F1Fo-ATP synthase. Here, we studied purified F1 from the beta R398C and beta R398W mutants. ATPase activity in both cases was resistant to aurovertin inhibition. The fluorescence spectrum (lambda exc = 278 or 295 nm) of beta R398W F1 showed a significant red-shift as compared to wild-type and beta R398C enzymes, indicating that residue beta R398 lies in a polar environment. On the basis of this and previous evidence, we propose that aurovertin binding to F1-ATPase involves a specific charged donor-acceptor H-bond between residue beta R398 and the 7-hydroxyl group of aurovertin. (2) The fluorescent substrate analog lin-benzo-ADP was shown to bind to beta R398W F1 catalytic sites with the same Kd values as to wild-type F1, and with the same quenching of the fluorescence of the analog. Fluorescence energy transfer was seen between the beta R398W residue and bound lin-benzo-ADP. Analysis of transfer efficiency at varying stoichiometry of bound lin-benzo-ADP showed that interaction occurred between one beta R398W residue and one catalytic-site-bound analog molecule at a distance of approximately 23 A. The relationships of the aurovertin and catalytic sites in the primary and tertiary structure are discussed.
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PMID:Investigation of the aurovertin binding site of Escherichia coli F1-ATPase by fluorescence spectroscopy and site-directed mutagenesis. 153 96


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