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 segment R165-T330 of the alpha subunit of Schizosaccharomyces pombe F1-ATPase, corresponding to a putative nucleotide-binding domain by comparison with related nucleotide-binding proteins, has been overexpressed in Escherichia coli. Produced as a nonsoluble material, it was purified in a nonnative form, using a rapid procedure that includes one reversed-phase chromatography step. Refolding of the domain, called DN alpha 19, was achieved quantitatively by using a high-dilution step and monitored by circular dichroism and intrinsic fluorescence. Once folded, DN alpha 19 was highly soluble and stable. It bound 1 mol/mol either of adenine or guanine di- or triphosphate nucleotide, with a Kd ranging from 2.3 to 5.4 microM, or of methylanthraniloyl derivatives of the same nucleotides, with a Kd ranging from 0.2 to 0.6 microM. Interesting, DN alpha 19 was able to hydrolyze nucleoside triphosphates at a low but significant rate. The distance between one tryptophan residue located in the nucleotide-binding site and the ribose-linked methylanthraniloyl group of di- or triphosphate nucleotides was estimated by fluorescence resonance energy transfer to be 13 or 11 A, respectively, suggesting that the tryptophan is close to the polyphosphate moiety of the nucleotide. This tryptophan residue was tentatively assigned to W190 by a hydrophobic cluster comparison with the H-ras p21 protein, suggesting that the putative loop of DN alpha 19 containing W190 could play a functional role in nucleotide binding.
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PMID:Functional nucleotide-binding domain in the F0F1-ATPsynthase alpha subunit from the yeast Schizosaccharomyces pombe. 839 82

Chemical modification of mitochondrial F1-ATPase from Schizosaccharomyces pombe by the tryptophan-specific reagent N-bromosuccinimide (NBS) at pH 5.0 in the presence of 20% glycerol produced a characteristic lowering in both enzyme absorbance at 280 nm and intrinsic fluorescence at 332 nm that varied with NBS/F1 molar ratio up to a value of 130. Fluorometric titration of tryptophans and correlation to residual ATPase activity showed that modification of three reactive residues among the seven present on alpha- and epsilon-subunits did not markedly modify the enzyme activity but efficiently released endogenous ATP and abolished the fluorescence quenching related to GDP or ATP binding to the catalytic site. Additional modification of one, less reactive, tryptophan altered both negative cooperativity of ATP hydrolysis and sensitivity to azide inhibition and produced a nearly complete inactivation at high NBS/F1 molar ratio. NBS-induced inactivation of F1 was favored by catalytic-site saturation with GDP or low ATP concentration and on the contrary was prevented by noncatalytic-site saturation with ADP or high ATP concentration. When reactive tryptophans were selectively modified by NBS in the presence of ADP, and subunits were isolated after guanidine hydrochloride dissociation by one-step purification on reversed-phase HPLC, the absorbance of alpha-subunit at 280 nm was decreased, whereas that of epsilon-subunit was unchanged. Cyanogen bromide cleavage of alpha-subunit and fragments separation by reversed-phase HPLC showed that one peptide of 3 kDa apparent molecular mass had decreased absorbance. N-Terminal sequencing allowed its identification to fragment 255-282 that contains tryptophan257.
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PMID:Chemical modification of alpha-subunit tryptophan residues in Schizosaccharomyces pombe mitochondrial F1 adenosine 5'-triphosphatase: differential reactivity and role in activity. 842 30

It had previously been suggested that Vmax hydrolysis rate of 2', 3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) by F1-ATPase required filling of only two catalytic sites on the enzyme (Grubmeyer, C., and Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727), whereas recently it was shown that Vmax rate of ATP hydrolysis requires that all three catalytic sites are filled (Weber, J., Wilke-Mounts, S., Lee, R. S. F., Grell, E., and Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133). To resolve this apparent discrepancy, we measured equilibrium binding and hydrolysis of MgTNP-ATP under identical conditions, using betaY331W mutant Escherichia coli F1-ATPase, in which the genetically engineered tryptophan provides a direct fluorescent probe of catalytic site occupancy. We found that MgTNP-ATP hydrolysis at Vmax rate did require filling of all three catalytic sites, but in contrast to the situation with MgATP, "bisite hydrolysis" of MgTNP-ATP amounted to a substantial fraction (approximately 40%) of Vmax. Binding of MgTNP-ATP to the three catalytic sites showed strong binding cooperativity (Kd1 < 1 nm, Kd2 = 23 nm, Kd3 = 1.4 microM). Free TNP-ATP (i.e. in presence of EDTA) bound to all three catalytic sites with lower affinity but was not hydrolyzed. These data emphasize that the presence of Mg2+ is critical for cooperativity of substrate binding, formation of the very high affinity first catalytic site, and hydrolytic activity in F1-ATPases and that these three properties are strongly correlated.
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PMID:Binding and hydrolysis of TNP-ATP by Escherichia coli F1-ATPase. 863 50

Using strategically-placed tryptophan (Trp) residues as optical probes to monitor nucleotide binding and hydrolysis, we demonstrate that all three catalytic nucleotide binding sites in F1-ATPase must be filled to obtain physiological (Vmax) MgATP hydrolysis rates. At Vmax hydrolysis rates, the predominant enzyme species has one of the three catalytic sites filled with unhydrolyzed substrate MgATP, the other two sites are filled with product MgADP. A specifically-inserted Trp probe was also developed to characterize nucleotide binding to the noncatalytic sites, and a model to explain the specificity of these sites is shown. These sites appear to play no role in ATP hydrolysis.
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PMID:F1F0-ATP synthase: development of direct optical probes of the catalytic mechanism. 868 41

Tryptophan was specifically inserted as the residue immediately preceding the P-loop sequence in F1-ATPase catalytic sites. The mutant enzyme (betaF148W) showed normal enzymatic characteristics. The fluorescence responses of beta-tryptophan 148 enabled us to differentiate between nucleoside di- and triphosphate bound in catalytic sites; MgADP quenched at 350 nm, whereas MgAMPPNP and MgADP.BeFx complex enhanced the fluorescence at 325 nm. With MgATP, both effects were seen simultaneously. This allowed analysis of bound catalytic site nucleotides directly under steady-state MgATP hydrolysis conditions. At mM concentration of MgATP (Vmax conditions) one of the three catalytic sites was filled with substrate MgATP and the other two sites were filled with product MgADP. A model for F1-ATPase steady-state turnover is presented that encompasses these findings. Given the structural similarity of the P-loop in nucleotide-binding proteins, this approach may prove widely useful.
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PMID:Specific tryptophan substitution in catalytic sites of Escherichia coli F1-ATPase allows differentiation between bound substrate ATP and product ADP in steady-state catalysis. 870 26

Using site-directed tryptophan fluorescence we studied nucleotide occupancy of the catalytic sites of Escherichia coli F1-ATPase, under conditions used previously for crystallization and X-ray structure analysis of the bovine mitochondrial enzyme [Abrahams et al. (1994) Nature 370, 621-628]. We found that only two of the three catalytic sites were filled in the E. coli enzyme under these conditions (250 microM MgAMPPNP plus 5 microM MgADP), consistent with what was reported in the bovine F1 X-ray structure. However, subsequent addition of a physiological concentration of MgATP readily filled the third catalytic site. Therefore the enzyme form seen in the X-ray structure results from the fact that it is obtained under sub-saturating nucleotide conditions. The data show that the X-ray structure is compatible with a catalytic mechanism in which all three F1-ATPase catalytic sites must fill with MgATP to initiate steady-state hydrolysis [e.g. Weber and Senior (1996) Biochim. Biophys. Acta 1275, 101-104]. The data further demonstrate that the site-directed tryptophan fluorescence technique can provide valuable support for F1 crystallography studies.
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PMID:Nucleotide occupancy of F1-ATPase catalytic sites under crystallization conditions. 907 28

Phosphorescence and fluorescence energy transfer measurements have been used to locate the epsilon-subunit within the know structural frame of the mitochondrial soluble part of F-type H(+)-ATPase complex (F1). The fluorescence probe 2'-O-(trinitrophenyl)adenosine-5'-triphosphate was bound to the nucleotide binding sites of the enzyme, whereas the probe 7-diethylamino-3'-(4'-maleimidylphenyl)-4-methylcoumarin was attached to the single sulfhydryl residue of isolated oligomycin sensitivity-conferring protein (OSCP), which was then reconstituted with F1. Fluorescence and phosphorescence resonance energy transfer yields from the lone tryptophan residue of F1 present in the epsilon-polypeptide and the fluorescence labels attached to the F1 complex established that tryptophan is separated by 3.7 nm from Cys-118 of OSCP in the reconstituted OSCP-F1 complex, by 4.9 nm from its closest catalytic site and by more than 6.4 nm from the two other catalytic sites, including the lowest affinity ATP site. These separations together with the crystallographic coordinates of the F1 complex (Abrahams, J.P., A. G. W. Leslie, R. Lutter, and J.E. Walker. 1994. Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature. 370:621-628) place the epsilon-subunit in the stem region of the F1 molecule in a unique asymmetrical position relative to the catalytic sites of the enzyme.
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PMID:Structural mapping of the epsilon-subunit of mitochondrial H(+)-ATPase complex (F1). 908 86

Using site-directed-tryptophan fluorescence, parameters for equilibrium binding of (Mg)TNP-ATP and (Mg)TNP-ADP to non-catalytic sites of Escherichia coli F1-ATPase were determined. All three non-catalytic sites showed the same affinity for MgTNP-ATP (Kd = 0.2 microM) or MgTNP-ADP (Kd = 6.5 microM) whereas even at concentrations of 100 microM no binding of uncomplexed TNP-ATP or TNP-ADP was observed. The results demonstrate that the three non-catalytic sites bind TNP-nucleotides non-cooperatively, and emphasize the importance of Mg2+ for non-catalytic-site nucleotide binding. Parameters for binding of (Mg)TNP-ADP to the three catalytic sites were also determined, and showed marked cooperativity. This work completes the set of thermodynamic parameters for equilibrium binding of (Mg)TNP-ATP and (Mg)TNP-ADP to all six nucleotide sites of F1, providing essential information to fully exploit the potential of these nucleotide analogs in studies of F1-ATPase.
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PMID:Binding of TNP-ATP and TNP-ADP to the non-catalytic sites of Escherichia coli F1-ATPase. 925 14

Coordination of the Mg2+ ion in Mg-nucleotide substrates by amino acid residue side chains in the catalytic site of Escherichia coli F1-ATPase was investigated. From the X-ray structure of the mitochondrial enzyme [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628], it may be inferred that the hydroxyl of betaThr-156 is a direct ligand of Mg2+, whereas the carboxyls of betaGlu-181, betaGlu-185, and betaAsp-242 might contribute via intervening water molecules. Elimination of each respective functional group by site-directed mutagenesis, followed by determination of Mg-nucleotide and uncomplexed nucleotide binding affinities using a tryptophan probe, showed that betaThr-156, betaGlu-185, and betaAsp-242 are all involved in Mg2+ coordination, whereas betaGlu-181 is not. A derived structural model for the octahedral coordination around the Mg2+ ion is presented. The results indicate that the ADP-containing site in the X-ray structure is the catalytic site of highest affinity. Correct Mg2+ coordination is required for catalytic activity at physiological rates. Elimination of any one of the Mg2+-coordinating residues led to complete loss of Mg2+-dependent nucleotide binding cooperativity of the catalytic sites.
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PMID:Mg2+ coordination in catalytic sites of F1-ATPase. 942 83

Novel tryptophan substitutions, surrounding the nucleotide bound in catalytic sites, were introduced into Escherichia coli F1-ATPase. The mutant enzymes were purified and studied by fluorescence spectroscopy. One cluster of Trp substitutions, consisting of beta-Trp-404, beta-Trp-410, beta-Asp-158 (lining the adenine-binding pocket), and beta-Trp-153 (close to the alpha/beta-phosphates), showed the same fluorescence responses to MgADP, MgAMPPNP, and MgATP and the same nucleotide binding pattern with MgADP and MgAMPPNP, with one site of higher and two sites of lower affinity. Therefore, in absence of catalytic turnover (and of gamma-subunit rotation), sites 2 and 3 appeared similar in affinity, and the region of the catalytic site sensed by these Trp substitutions did not change conformation with different nucleotides. In contrast, alpha-Trp-291 and beta-Trp-297, both close to the gamma-phosphate, showed very different fluorescence responses to MgADP versus MgAMPPNP, and in these cases the response was due exclusively or predominantly to nucleotide binding at the first, high-affinity catalytic site, thus allowing specific detection of this site. Titration with MgATP showed that the high-affinity site was present under conditions of steady-state, Vmax MgATP hydrolysis.
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PMID:Tryptophan substitutions surrounding the nucleotide in catalytic sites of F1-ATPase. 972 15


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