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)

Atomic absorption and electron paramagnetic resonance spectroscopy were used to study the metal binding sites of beef heart mitochondrial ATPase (F1). Quantitative and qualitative properties of these sites are described. Two different separation techniques were able to distinguish two very tight sites from one tight (easily exchangeable) metal binding site on F1. Of these sites, two are specific for magnesium while one can be substituted with Mn2+, Co2+, or Zn2+. When MgAMP-PNP was incubated with F1, a fourth metal was bound to the enzyme. The carboxyl group modified by dicyclohexylcarbodiimide is shown not to be involved in binding of any of the tightly bound metals. Qualitative properties of the metal binding sites using the Mn2+-enzyme complex as a probe were ascertained using EPR at pH 6.8 and 8.0. CrATP and Mn2+ appear to bind to different metal sites on F1. The possible role of the metals in regulation of catalysis, and their relation to nucleotide binding is discussed.
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PMID:Metal interactions with beef heart mitochondrial ATPase. 286 Jan 5

We have identified the most probable protein ligands at the catalytic M3 and noncatalytic M2 metal-binding sites in the spinach chloroplast F1-ATPase (CF1) and here propose possible residues in the protein sequence for these ligands in latent CF1 in the absence of nucleotide. The changes in the metal ligands at these sites upon binding of nucleotide to the N2 and N3 sites and upon activation of latent CF1 provide a possible molecular basis for inhibition of ATPase activity by free metal, for the lack of activity in the latent state, and for the gating mechanism of the ATPase H+ pump. To these ends, the Mg2+ analogue, vanadyl (VIV = O)2+, was used as a paramagnetic probe at the M2 and M3 metal-binding sites. EPR and ESEEM spectra of VO2+ were obtained, and simulations of the full EPR spectra imply the ligand sets at the different metal-binding sites. When VO2+ is added to CF1 in the absence of ATP, the most likely set of ligands at the M2 site are 1 ROH (alpha T176), 2 H2O, and 1 RCOO- (alpha D269 or alpha D270), where the suggested amino acid designations of the residues are given in parentheses according to the mitochondrial sequence. Evidence suggests a possible axial nitrogen ligand at this site (alpha K175). When the M2 site is filled by addition of VO2+ and ATP, the metal binds as a second species in which N2-bound ATP and M2-bound VO2+ form a monodentate complex with concomitant exchange of the equatorial protein ligands by 3 H2O.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of nucleotides on the protein ligands to metals at the M2 and M3 metal-binding sites of the spinach chloroplast F1-ATPase. 788 Aug 23

Vanadyl (VIV=O)2+ was used as a paramagnetic probe at the M2 and M3 metal-binding sites of the spinach chloroplast F1-ATPase (CF1) in order to detect interaction of the metals with nucleotides. The M2 site can exist in two forms in the presence of ATP. When ATP and VO2+ are added in a 1.5:1 ratio to CF1, the VO2+ EPR spectrum is identical to that of CF1-VO2+ in the absence of ATP. When the M2 site is filled by the addition of ATP and VO2+ in a 3:1 ratio, the VO2+ binds to M2 in a second form with equatorial coordination to a single phosphate. The treatments required to deplete CF1 of the monodentate VO2+(-)nucleotide complex indicate that the VO2+ is coordinated to the ATP at the nonacatalytic N2 site. The presence of uncomplexed nucleotide appears to induce formation of the second form, possibly via ATP binding to the N3 site. This change in coordination at the M2 noncatalytic site may regulate the ATPase activity of CF1. The M3 site also exists in two forms: (i) in latent CF1, no phosphate coordination is evident; and (ii) after the ATPase has been activated, the EPR line shape is consistent with the two phosphates from ADP at N3 coordinated to the VO2+ at M3. This work establishes a connection between the metal- and nucleotide-binding sites as M2-N2 and M3-N3.
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PMID:Coordination of nucleotides to metals at the M2 and M3 metal-binding sites of spinach chloroplast F1-ATPase. 806 Sep 67

Vanadyl, (V = O)2+, is able to substitute for Mg2+ as a cofactor for ATPase activity catalyzed by the chloroplast F1-ATPase (CF1). Mg2+-dependent ATPase activity was also observed with CF1 that contained VO(2+)-ATP bound specifically to the noncatalytic N2 site. Modulation of the Mg(2+)-ATPase activity induced by VO2+ bound at this site indicates that the metal bound to the noncatalytic site affects catalytic activity. When CF1 is depleted of nucleotides from all but the N1 site, a single Mg2+ remains bound at a site designated M1. Addition of VO2+ to the depleted protein gives rise to an EPR spectrum characteristic of a CF1-bound VO2+ species. The binding curve of the VO2+ complex to latent, nucleotide-depleted CF1 was determined by the integrated intensities of the -5/2 parallel peak in the EPR spectrum as calibrated using atomic absorption spectroscopy. Under these conditions, VO2+ binds cooperatively to approximately two sites designated M2 and M3. Three-pulse ESEEM spectra of the CF1-VO2+ complex contain two intense modulations with frequencies and field-dependent behavior that show that they are from a directly coordinated 14N nucleus. Analysis of the bound VO2+ by ENDOR spectroscopy revealed the presence of a single group of protons associated with an equatorial amino or water ligand that is exchangeable with solvent. Using the additivity relation for hyperfine coupling, the most probable set of equatorial ligands to the VO2+ bound to CF1 under these conditions consists of one lysine nitrogen, two carboxyl oxygens from aspartate or glutamate, and one water.
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PMID:Characterization of ligands of a high-affinity metal-binding site in the latent chloroplast F1-ATPase by EPR spectroscopy of bound VO2+. 816 51

The mutation E204Q in the beta subunit of the chloroplast F1-ATPase was made by biolistic transformation of Chlamydomonas reinhardtii. The yield of chloroplast F1-ATPase (CF1) purified from thylakoids was unaltered, suggesting that the mutation did not affect protein assembly. However, photoautotrophic growth of Chlamydomonas strains containing beta E204Q was virtually abolished, and the effect of the mutation on the light-driven ATPsynthase activity catalyzed by purified thylakoids was comparable to the change in the photoautotrophic growth rate. The loss of ATPsynthase activity in the mutant was not the result of uncoupling. Addition of wild-type CF1 to mutant thylakoids depleted of CF1 reconstituted ATPsynthase activity indicating that the mutation did not affect assembly of F0. Furthermore, the mutant CF1F0 was capable of catalyzing ATPase-dependent proton pumping as measured by fluorescence quenching of 9-amino acridine. Although the mutation significantly affected the apparent kcat/K(m) of the Mg(2+)-ATPase activity of the purified CF1-ATPase, no significant effect on the apparent kcat was observed with the mutant compared to wild-type. No significant changes in the ability of Mg2+ or Mn2+ to serve either as a cofactor or as an inhibitor of ATPase activity were observed in the mutants relative to the wild-type CF1-ATPase. EPR spectra were also taken of VO2+ bound at catalytic site 3 in its latent form. In a large fraction of the latent enzyme, a carboxyl group has displaced the nucleotide-phosphate coordination to the metal which results in the free-metal inhibited form (M3). No significant effects on the gII and AII 51V hyperfine parameters were observed between wild-type and mutant. However, the mutation increased the abundance of the M3 form relative to the M3-N3 form (metal-nucleotide-coordinated form). On the basis of these results, beta E204 is not the carboxyl group that displaces the nucleotide phosphate as a ligand to form the free-metal inhibited enzyme form which predominates in site 3 in the latent state. Instead, the data are consistent with a role in which beta E204 is essential to protonate an inorganic phosphate-oxygen to make that oxygen a good leaving group to facilitate ATP synthesis and, via this role in H-bonding, increases the abundance of the functional metal-nucleotide complex bound to the catalytic site.
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PMID:Catalytic and EPR studies of the beta E204Q mutant of the chloroplast F1-ATPase from Chlamydomonas reinhardtii. 881 Sep 28

Site-directed mutations were made to the phosphate-binding loop threonine in the beta-subunit of the chloroplast F1-ATPase in Chlamydomonas (betaT168). Rates of photophosphorylation and ATPase-driven proton translocation measured in coupled thylakoids purified from betaT168D, betaT168C, and betaT168L mutants had <10% of the wild type rates, as did rates of Mg2+-ATPase activity of purified chloroplast F1-ATPase (CF1). The EPR spectra of VO2+-ATP bound to Site 3 of CF1 from wild type and mutants showed that EPR species C, formed exclusively upon activation, was altered in CF1 from each mutant in both signal intensity and in 51V hyperfine parameters that depend on the equatorial VO2+ ligands. These data provide the first direct evidence that Site 3 is a catalytic site. No significant differences between wild type and mutants were observed in EPR species B, the predominant form of the latent enzyme. Thus, the phosphate-binding loop threonine is an equatorial metal ligand in the activated conformation but not in the latent conformation of Site 3. The metal-nucleotide conformation that gives rise to species B is consistent with the Mg2+-ADP complex that becomes entrapped in a catalytic site in a manner that regulates enzymatic activity. The lack of catalytic function of CF1 with entrapped Mg2+-ADP may be explained in part by the absence of the phosphate-binding loop threonine as a metal ligand.
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PMID:EPR spectroscopy of VO2+-ATP bound to catalytic site 3 of chloroplast F1-ATPase from Chlamydomonas reveals changes in metal ligation resulting from mutations to the phosphate-binding loop threonine (betaT168). 1006 66

The Mg(2+) cofactor of the F(1)F(0) ATP synthase is required for the asymmetry of the catalytic sites that leads to the differences in affinity for nucleotides. Vanadyl (V(IV)=O)(2+) is a functional surrogate for Mg(2+) in the F(1)-ATPase. The (51)V-hyperfine parameters derived from EPR spectra of VO(2+) bound to specific sites on the enzyme provide a direct probe of the metal ligands at each site. Site-directed mutations of residues that serve as metal ligands were found to cause measurable changes in the (51)V-hyperfine parameters of the bound VO(2+), thereby providing a means by which metal ligands were identified in the functional enzyme in several conformations. At the low-affinity catalytic site comparable to beta(E) in mitochondrial F(1), activation of the chloroplast F(1)-ATPase activity induces a conformational change that inserts the P-loop threonine and catch-loop tyrosine hydroxyl groups into the metal coordination sphere thereby displacing an amino group and the Walker homology B aspartate. Kinetic evidence suggests that coordination of this tyrosine by the metal when the empty site binds substrate may provide an escapement mechanism that allows the gamma subunit to rotate and the conformation of the catalytic sites to change, thereby allowing rotation only when the catalytic sites are filled. In the high-affinity conformation analogous to the beta(DP) site of mitochondrial F(1), the catch-loop tyrosine has been displaced by carboxyl groups from the Walker homology B aspartate and from betaE197 in Chlamydomonas CF(1). Coordination of the metal by these carboxyl groups contributes significantly to the ability of the enzyme to bind the nucleotide with high affinity.
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PMID:The participation of metals in the mechanism of the F(1)-ATPase. 1083 47

The arrangement of the b-subunits in the holo-enzyme F(0)F(1)-ATP synthase from E. coli is investigated by site-directed mutagenesis spin-label EPR. F(0)F(1)-ATP synthases couple proton translocation with the synthesis of ATP from ADP and phosphate. The hydrophilic F(1)-part and the hydrophobic membrane-integrated F(0)-part are connected by a central and a peripheral stalk. The peripheral stalk consists of two b-subunits. Cysteine mutations are introduced in the tether domain of the b-subunit at b-40, b-51, b-53, b-62 or b-64 and labeled with a nitroxide spin label. Conventional (9 GHz), high-field (95 GHz) and pulsed EPR spectroscopy reveal: All residues are in a relatively polar environment, with mobilities consistent with helix sites. The distance between the spin labels at each b-subunit is 2.9 nm in each mutant, revealing a parallel arrangement of the two helices. They can be in-register but separated by a large distance (1.9 nm), or at close contact and displaced along the helix axes by maximally 2.7 nm, which excludes an in-register coiled-coil model suggested previously for the b-subunit. Binding of the non-hydrolysable nucleotide AMPPNP to the spin-labeled enzyme had no significant influence on the distances compared to that in the absence of nucleotides.
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PMID:Distances between the b-subunits in the tether domain of F(0)F(1)-ATP synthase from E. coli. 1590 87

Plant chloroplasts are particularly threatened by free radical attack. We incubated purified soluble spinach chloroplast F(0)F(1) (CF(0)F(1), EC 3.6.3.34) with an Fe(2+)/H(2)O(2)/ascorbate system, and about 60% inactivation of the ATPase activity was reached after 60 min. Inactivation was not prevented by omission of H(2)O(2), by addition of catalase or superoxide dismutase, nor by the scavengers mannitol, DMSO, or BHT. No evidence for enzyme fragmentation or oligomerization was detected by SDS-PAGE. The chloroplast ATP synthase is resistant to attack by the reactive oxygen species commonly found at the chloroplast level. DTT in the medium completely prevented the inhibition, and its addition after the inhibition partially recovered the activity of the enzyme. CF(0)F(1) thiol residues were lost upon oxidation. The rate of thiol modification was faster than the rate of enzyme inactivation, suggesting that the thiol residues accounting for the inhibition may be hindered. Enzyme previously oxidized by iodobenzoate was not further inhibited by the oxidative system. The production of ascorbyl radical was identified by EPR and is possibly related to CF(0)F(1) inactivation. It is thus suggested that the ascorbyl radical, which accumulates under plant stress, might regulate CF(0)F(1).
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PMID:Inhibition of spinach chloroplast F0F1 by an Fe2+/ascorbate/H2O2 system. 1787 May 88

Dynamic nuclear polarization is emerging as a potential tool to increase the sensitivity of NMR aiming at the detection of macromolecules in liquid solution. One possibility for such an experimental design is to perform the polarization step between electrons and nuclei at low magnetic fields and then transfer the sample to a higher field for NMR detection. In this case, an independent optimization of the polarizer and detection set ups is required. In the present paper we describe the optimization of a polarizer set up at 15 MHz (1)H NMR/9.7 GHz EPR frequencies based on commercial hardware. The sample consists of the nitroxide radical TEMPONE-D,(15)N in water, for which the dimensions were systematically decreased to fit the homogeneous B(1) region of a dielectric ENDOR resonator. With an available B(1) microwave field up to 13 G we observe a maximum DNP enhancement of -170 at room temperature by irradiating on either one of the EPR lines. The DNP enhancement was saturated at all polarizer concentrations. Pulsed ELDOR experiments revealed that the saturation level of the two hyperfine lines is such that the DNP enhancements are well consistent with the coupling factors derived from NMRD data. By raising the polarizing field and frequencies 10-fold, i.e. to 140 MHz (1)H/94 GHz EPR, we reach an enhancement of -43 at microwave field strengths (B(1) approximately 5 G). The results are discussed in view of an application for a DNP spectrometer.
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PMID:Optimization of dynamic nuclear polarization experiments in aqueous solution at 15 MHz/9.7 GHz: a comparative study with DNP at 140 MHz/94 GHz. 2045 34


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