ATP synthase. Conditions under which all catalytic sites of the F1 moiety are kinetically equivalent in hydrolyzing ATP

Conditions have been reported under which the F1 moiety of bovine heart ATP synthase catalyzes the hydrolysis of ATP by an apparently cooperative mechanism in which the slow rate of hydrolysis at a single catalytic site (unisite catalysis) is enhanced more than 10(6)-fold when ATP is added in excess to occupy one or both of the other two catalytic sites (multisite catalysis) (Cross, R. L., Grubmeyer, C., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12101-12105). In the novel studies reported here, and in contrast to the earlier report, we have (a) monitored the kinetics of ATP hydrolysis of F1 by using nucleotide-depleted preparations and a highly sensitive chemiluminescent assay; (b) followed the reaction immediately upon addition of F1 to ATP, rather than after prior incubation with ATP; and (c) used a reaction medium with Pi as the only buffer. The following observations were noted. First, regardless of the source of enzyme, bovine or rat, and catalytic conditions (unisite or multisite), the rates of hydrolysis depend on ATP concentration to the first power. Second, the first order rate constant for ATP hydrolysis remains relatively constant under both unisite and multisite conditions declining only slightly at high ATP concentration. Third, the initial rates of ATP hydrolysis exhibit Michaelis-Menten kinetic behavior with a single Vmax exceeding 100 micromol of ATP hydrolyzed per min/mg of F1 (turnover number = 635 s-1) and a single Km for ATP of about 57 microM. Finally, the reaction is inhibited markedly by low concentrations of ADP. It is concluded that, under the conditions described here, all catalytic sites that participate in the hydrolysis of ATP within the F1 moiety of mitochondrial ATP synthase function in a kinetically equivalent manner.     

An epidemiologic study on the aetiological factors of primary liver cancer in Shunde City of Guangdong province

The F-type ATPase, TF0F1, from the thermophilic Bacillus PS3, which is free of nucleotides after isolation, was specifically loaded with one 2-azido ADP on a non-catalytic site. The enzyme was covalently modified to various extents and the rate of ATP synthesis and ATP hydrolysis was measured. Both ATP synthesis and ATP hydrolysis extrapolated to zero for covalently binding one nucleotide per enzyme. This was interpreted such that the non-catalytic sites are involved in the coupled catalytic process.     

Conformational changes of the mitochondrial F1-ATPase epsilon-subunit induced by nucleotide binding as observed by phosphorescence spectroscopy

Changes in conformation of the epsilon-subunit of the bovine heart mitochondrial F1-ATPase complex as a result of nucleotide binding have been demonstrated from the phosphorescence emission of tryptophan. The triplet state lifetime shows that whereas nucleoside triphosphate binding to the enzyme in the presence of Mg2+ increases the flexibility of the protein structure surrounding the chromophore, nucleoside diphosphate acts in an opposite manner, enhancing the rigidity of this region of the macromolecule. Such changes in dynamic structure of the epsilon-subunit are evident at high ligand concentration added to both the nucleotide-depleted F1 (Nd-F1) and the F1 preparation containing the three tightly bound nucleotides (F1(2,1)). Since the effects observed are similar in both the F1 forms, the binding to the low affinity sites must be responsible for the conformational changes induced in the epsilon-subunit. This is partially supported by the observation that the Trp lifetime is not significantly affected by adding an equimolar concentration of adenine nucleotide to Nd-F1. The effects on protein structure of nucleotide binding to either catalytic or noncatalytic sites have been distinguished by studying the phosphorescence emission of the F1 complex prepared with the three noncatalytic sites filled and the three catalytic sites vacant (F1(3,0)). Phosphorescence lifetime measurements on this F1 form demonstrate that the binding of Mg-NTP to catalytic sites induces a slight enhancement of the rigidity of the epsilon-subunit. This implies that the binding to the vacant noncatalytic site of F1(2,1) must exert the opposite and larger effect of enhancing the flexibility of the protein structure observed in both Nd-F1 and F1(2,1). The observation that enhanced flexibility of the protein occurs upon addition of adenine nucleotides to F1(2,1) in the absence of Mg2+ provides direct support for this suggestion. The connection between changes in structure and the possible functional role of the epsilon-subunit is discussed.     

Intersubunit rotation in active F-ATPase

The enzyme ATP synthase, or F-ATPase, is present in the membranes of bacteria, chloroplasts and mitochondria. Its structure is bipartite, with a proton-conducting, integral membrane portion, F0, and a peripheral portion, F1. Solubilized F1 is composed of five different subunits, (alpha beta)3 gamma delta epsilon, and is active as an ATPase. The function of F-ATPase is to couple proton translocation through F0 with ATP synthesis in F1 (ref.3). Several lines of evidence support the spontaneous formation of ATP on F1 (refs 4,5) and its endergonic release at cooperative and rotating (or at least alternating) sites. The release of ATP at the expense of protonmotive force might involve mechanical energy transduction from F0 into F1 by rotation of the smaller subunits (mainly gamma) within (alpha beta)3, the catalytic hexagon of F1 as suggested by electron microscopy, by X-ray crystal structure analysis and by the use of cleavable crosslinkers. Here we record an intersubunit rotation in real time in the functional enzyme by applying polarized absorption relaxation after photobleaching to immobilized F1 with eosin-labelled gamma. We observe the rotation of gamma relative to immobilized (alpha beta)3 in a timespan of 100 ms, compatible with the rate of ATP hydrolysis by immobilized F1. Its angular range, which is of at least 200 degrees, favours a triple-site mechanism of catalysis, with gamma acting as a crankshaft in (alpha beta)3. The rotation of gamma is blocked when ATP is substituted with its non-hydrolysable analogue AMP-PNP.     

Molecular distance measurements reveal an (alpha beta)2 dimeric structure of Na+/K+-ATPase. High affinity ATP binding site and K+-activated phosphatase reside on different alpha-subunits

ATP hydrolysis by Na+/K+-ATPase proceeds via the interaction of simultaneously existing and cooperating high (E1ATP) and low (E2ATP) substrate binding sites. It is unclear whether both ATP sites reside on the same or on different catalytic alpha-subunits. To answer this question, we looked for a fluorescent label for the E2ATP site that would be suitable for distance measurements by F?rster energy transfer after affinity labeling of the E1ATP site by fluorescein 5'-isothiocyanate (FITC). Erythrosin 5'-isothiocyanate (ErITC) inactivated, in an E1ATP site-blocked enzyme (by FITC), the residual activity of the E2ATP site, namely K+-activated p-nitrophenylphosphatase in a concentration-dependent way that was ATP-protectable. The molar ratios of FITC/alpha-subunit of 0.6 and of ErITC/alpha-subunit of 0.48 indicate 2 ATP sites per (alpha beta)2 diprotomer. Measurements of F?rster energy transfer between the FITC-labeled E1ATP and the ErITC-labeled or Co(NH3)4ATP-inactivated E2ATP sites gave a distance of 6.45 +/- 0.64 nm. This distance excludes 2 ATP sites per alpha-subunit since the diameter of alpha is 4-5 nm. F?rster energy transfer between cardiac glycoside binding sites labeled with anthroylouabain and fluoresceinylethylenediamino ouabain gave a distance of 4.9 +/- 0.5 nm. Hence all data are consistent with the hypothesis that Na+/K+-ATPase in cellular membranes is an (alpha beta)2 diprotomer and works as a functional dimer (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321).     

Modeling of nucleotide binding domains of ABC transporter proteins based on a F1-ATPase/recA topology: structural model of the nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR)

Members of the ABC transporter superfamily contain two nucleotide binding domains. To date, the three dimensional structure of no member of this super-family has been elucidated. To gain structural insight, the known structures of several other nucleotides binding proteins can be used as a framework for modeling these domains. We have modeled both nucleotide binding domains of the protein CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) using the two similar domains of mitochondrial F1-ATPase. The models obtained, provide useful insights into the putative functions of these domains and their possible interaction as well as a rationale for the basis of Cystic Fibrosis causing mutations. First, the two nucleotide binding domains (folds) of CFTR are each predicted to span a 240-250 amino acid sequence rather than the 150-160 amino acid sequence originally proposed. Second, the first nucleotide binding fold, is predicted to catalyze significant rates of ATP hydrolysis as a catalytic base (E504) resides near the y phosphate of ATP. This prediction has been verified experimentally [Ko, Y.H., and Pedersen, P.L. (1995) J. Biol. Chem. 268, 24330-24338], providing support for the model. In contrast, the second nucleotide binding fold is predicted at best to be a weak ATPase as the glutamic acid residue is replaced with a glutamine. Third, F508, which when deleted causes approximately 70% of all cases of cystic fibrosis, is predicted to lie in a cleft near the nucleotide binding pocket. All other disease causing mutations within the two nucleotide binding domains of CFTR either reside near the Walker A and Walker B consensus motifs in the heart of the nucleotide binding pocket, or in the C motif which lies outside but near the nucleotide binding pocket. Finally, the two nucleotide binding domains of CFTR are predicted to interact, and in one of the two predicted orientations, F508 resides near the interface. This is the first report where both nucleotide binding domains of an ABC transporter and their putative domain-domain interactions have been modeled in three dimensions. The methods and the template used in this work can be used to analyze the structures and function of the nucleotide binding domains of all other members of the ABC transporter super-family.     

Effects of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin on nucleotide binding to the three F1-ATPase catalytic sites measured using specific tryptophan probes

Equilibrium nucleotide binding to the three catalytic sites of Escherichia coli F1-ATPase was measured in the presence of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin to elucidate mechanisms of inhibition. Fluorescence signals of beta-Trp-331 and beta-Trp-148 substituted in catalytic sites were used to determine nucleotide binding parameters. Azide brought about small decreases in Kd(MgATP) and Kd(MgADP). Notably, under MgATP hydrolysis conditions, it caused all enzyme molecules to assume a state with three catalytic site-bound MgATP and zero bound MgADP. These results rule out the idea that azide inhibits by "trapping" MgADP. Rather, azide blocks the step at which signal transmission between catalytic sites promotes multisite hydrolysis. Aurovertin bound with stoichiometry of 1.8 (mol/mol of F1) and allowed significant residual turnover. Cycling of the aurovertin-free beta-subunit catalytic site through three normal conformations was indicated by MgATP binding data. Aurovertin did not change the normal ratio of 1 bound MgATP/2 bound MgADP in catalytic sites. The results indicate that it acts to slow the switch of catalytic site affinities ("binding change step") subsequent to MgATP hydrolysis. Dicyclohexylcarbodiimide shifted the ratio of catalytic site-bound MgATP/MgADP from 1:2 to 1.6:1.4, without affecting Kd(MgATP) values. Like azide, it also appears to affect activity at the step after MgATP binding, in which signal transmission between catalytic sites promotes MgATP hydrolysis.     

Energy transduction in ATP synthase

Mitochondria, bacteria and chloroplasts use the free energy stored in transmembrane ion gradients to manufacture ATP by the action of ATP synthase. This enzyme consists of two principal domains. The asymmetric membrane-spanning F0 portion contains the proton channel, and the soluble F1 portion contains three catalytic sites which cooperate in the synthetic reactions. The flow of protons through F0 is thought to generate a torque which is transmitted to F1 by an asymmetric shaft, the coiled-coil gamma-subunit. This acts as a rotating 'cam' within F1, sequentially releasing ATPs from the three active sites. The free-energy difference across the inner membrane of mitochondria and bacteria is sufficient to produce three ATPs per twelve protons passing through the motor. It has been suggested that this proton motive force biases the rotor's diffusion so that F0 constitutes a rotary motor turning the gamma shaft. Here we show that biased diffusion, augmented by electrostatic forces, does indeed generate sufficient torque to account for ATP production. Moreover, the motor's reversibility-supplying torque from ATP hydrolysis in F1 converts the motor into an efficient proton pump-can also be explained by our model.     

The Escherichia coli FOF1 gammaM23K uncoupling mutant has a higher K0.5 for Pi. Transition state analysis of this mutant and others reveals that synthesis and hydrolysis utilize the same kinetic pathway

The Escherichia coli FOF1 ATP synthase uncoupling mutation, gammaM23K, was found to increase the energy of interaction between gamma and beta subunits, prevent the proper utilization of binding energy to drive catalysis, and block the enzyme in a Pi release mode. In this paper, the effects of this mutation on substrate binding in cooperative ATP synthesis are assessed. Activation of ATP synthesis by ADP and Pi was determined for the gammaM23K FOF1. The K0.5 for ADP was not affected, but K0.5 for Pi was approximately 7-fold higher even though the apparent Vmax was close to the wild-type level. Wild-type enzyme had a turnover number of 82 s-1 at pH 7.5 and 30 degrees C. During oxidative phosphorylation, the apparent dissociation constant (KI) for ATP was not affected and was 5-6 mM for both wild-type and gammaM23K enzymes. Thus, the apparent binding affinity for ATP in the presence of DeltamuH+ was lowered by 7 orders of magnitude from the affinity measured at the high-affinity catalytic site. Arrhenius analysis of ATP synthesis for the gammaM23K FOF1 revealed that, like those of ATP hydrolysis, the transition state DeltaH was much more positive and TDeltaS was much less negative, adding up to little change in DeltaG. These results suggested that ATP synthesis is inefficient because of an extra bond between gamma and beta subunits which must be broken to achieve the transition state. Analysis of the transition state structures using isokinetic plots demonstrate that ATP hydrolysis and synthesis utilize the same kinetic pathway. Incorporating this information into a model for rotational catalysis suggests that at saturating substrate concentrations, the rate-limiting step for hydrolysis and synthesis is the rotational power stroke where each of the beta subunits changes conformation and affinity for nucleotide.     

Morphometric studies on the effects of dehydration on the renin-immunopositive cells in the kidney of the Mongolian gerbil (Meriones unguiculatus)

Labeling of mitochondrial F1-ATPase with 8-azido-ATP or 8-azido-ADP under turnover conditions with Mg(2+)-ATP resulted in the identification of one exchangeable non-catalytic site whose occupation with a ligand does not influence the ATPase activity of F1 when measured at Vmax. With 8-azido-ADP two exchangeable non-catalytic sites could be labeled, but at one of them the bound ligand exchanges, at least partly, during the illumination under turnover conditions. After labeling an exchangeable non-catalytic site under turnover conditions with 8-azido-ATP or with 8-azido-ADP, F1-ATPase kept the ability to bind NAP3-2N3ADP at the slowly exchangeable noncatalytic site, thereby inhibiting the ATPase activity by 45%, as recently described (Edel et al. (1992) Biochim. Biophys. Acta 1101, 329-338). Covalent modification of the low-affinity non-catalytic site with 8-nitreno-AT(D)P increased the Km of ATP and abolished the negative cooperativity of ATP hydrolysis. This site can therefore be marked as a regulatory site, whose occupation with a nucleotide decreases the affinity of the catalytic sites for ATP.     

A two-site mechanism for ATP hydrolysis by the asymmetric Rep dimer P2S as revealed by site-specific inhibition with ADP-A1F4

The Escherichia coli Rep helicase is a dimeric motor protein that catalyzes the transient unwinding of duplex DNA to form single-stranded (ss) DNA using energy derived from the binding and hydrolysis of ATP. In an effort to understand this mechanism of energy transduction, we have used pre-steady-state methods to study the kinetics of ATP binding and hydrolysis by an important intermediate in the DNA unwinding reaction--the asymmetric Rep dimer state, P2S, where ss DNA [dT(pT)15] is bound to only one subunit of the Rep dimer. To differentiate between the two potential ATPase active sites inherent in the dimer, we constructed dimers with one subunit covalently cross-linked to ss DNA and where one or the other of the ATPase sites was selectively complexed to the tightly bound transition state analog ADP-A1F4. We found that when ADP-A1F4 is bound to the Rep subunit in trans from the subunit bound to ss DNA, steady-state ATPase activity of 18 s(-1) per dimer (equivalent to wild-type P2S) was recovered. However, when the ADP-A1F4 and ss DNA are both bound to the same subunit (cis), then a titratable burst of ATP hydrolysis is observed corresponding to a single turnover of ATP. Rapid chemical quenched-flow techniques were used to resolve the following minimal mechanism for ATP hydrolysis by the unligated Rep subunit of the cis dimer: E + ATP <==> E-ATP <==> E'-ATP <==> E'-ADP-Pi <==> E-ADP-Pi <==> E-ADP + Pi <==> E + ADP + Pi, with K1 = (2.0 +/- 0.85) x 10(5) M(-1), k2 = 22 +/- 3.5 s(-1), k(-2) < 0.12 s(-1), K3 = 4.0 +/- 0.4 (k3 > 200 s(-1)), k4 = 1.2 +/- 0.14 s(-1), k(-4) < 1.2 s(-1), K5 = 1.0 +/- 0.2 mM, and K6 = 80 +/- 8 microM. A salient feature of this mechanism is the presence of a kinetically trapped long-lived tight nucleotide binding state, E'-ADP-Pi. In the context of our "subunit switching" model for Rep dimer translocation during processive DNA unwinding [Bjornson, K. B., Wong, I., & Lohman, T. M. (1996) J. Mol. Biol. 263, 411-422], this state may serve an energy storage function, allowing the energy from the binding and hydrolysis of ATP to be harnessed and held in reserve for DNA unwinding.     

The alpha 3(beta Y341W)3 gamma subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 fails to dissociate ADP when MgATP is hydrolyzed at a single catalytic site and attains maximal velocity when three catalytic sites are saturated with MgATP

The hydrolytic properties of the alpha3beta3gamma and mutant alpha3(betaY341W)3gamma subcomplexes of the TF1-ATPase have been compared. ATPase activity of the mutant is less sensitive to turnover-dependent inhibition by azide, less suppressed by increasing concentrations of Mg2+ during assay, and less stimulated by lauryl dimethylamine oxide (LDAO). Therefore, it has much lower propensity than wild-type to entrap inhibitory MgADP in a catalytic site during turnover. The fluorescence of the introduced tryptophans in the alpha3(betaY341W)3gamma subcomplex is completely quenched when catalytic sites are saturated with ATP or ADP with or without Mg2+ present. As reported for the betaY331W mutant of Escherichia coli F1 (Weber, J., Wilke-Mounts, S., Lee, R. S.-F., Grell, E., Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133), this provides a direct probe of nucleotide binding to catalytic sites. Addition of stoichiometric MgATP to the mutant subcomplex quenched one-third the tryptophan fluorescence which did not recover after 60 min. This was caused by entrapment of MgADP in a single catalytic site. Titration of catalytic sites of the alpha3(betaY341W)3gamma subcomplex with MgADP or MgATP revealed Kd's of < 50 nM, about 0.25 microM and about 35 microM. Titrations were not affected by azide, whereas LDAO lowered the affinities of catalytic sites 2 and 3 for MgADP by 5-fold and 2-fold, respectively. During titration with MgATP, LDAO slightly lowered affinity at ATP concentrations below 30 microM and had no effect at ATP concentrations above 30 microM. Maximal velocity was attained when the third catalytic site was titrated with MgATP in the presence or absence of LDAO. The same Kd's for binding MgATP to the (alphaA396C)3beta3(gammaA22C) mutant were observed before and after inactivating it by cross-linking alpha to gamma. This implies that the different affinities of catalytic sites for MgATP do not represent negative cooperativity, but rather represent heterogeneous affinities of catalytic sites dictated by the position of the coiled-coil of the gamma subunit within the central cavity of the (alpha beta)3 hexamer.     

A mutation in the Escherichia coli F0F1-ATP synthase rotor, gammaE208K, perturbs conformational coupling between transport and catalysis

Cross-linking studies on the Escherichia coli F0F1-ATP synthase indicated a site of interaction involving gamma and epsilon subunits in F1 and subunit c in F0 (Watts, S. D., Tang, C., and Capaldi, R. A. (1996) J. Biol. Chem. 271, 28341-28347). To assess the function of these interactions, we introduced random mutations in this region of the gamma subunit (gamma194-213). One mutation, gammaGlu-208 to Lys (gammaE208K), caused a temperature-sensitive defect in oxidative phosphorylation-dependent growth. ATP hydrolytic rates of the gammaE208K F0F1 enzyme became increasingly uncoupled from H+ pumping above 28 degreesC. In contrast, Arrhenius plot of steady-state ATP hydrolysis of the mutant enzyme was linear from 20 to 50 degreesC. Analysis of this plot revealed a significant increase in the activation energy of the catalytic transition state to a value very similar to soluble, epsilon subunit-inhibited F1 and suggested that the mutation blocked normal release of epsilon inhibition of ATP hydrolytic activity upon binding of F1 to F0. The difference in temperature dependence suggested that the gammaE208K mutation perturbed release of inhibition via a different mechanism than it did energy coupling. Suppressor mutations in the polar loop of subunit c restored ATP-dependent H+ pumping and transition state thermodynamic parameters close to wild-type values indicating that interactions between gamma and c subunits mediate release of epsilon inhibition and communication of coupling information.     

Nucleotide-promoted release of hMutSalpha from heteroduplex DNA is consistent with an ATP-dependent translocation mechanism

ATP hydrolysis by bacterial and eukaryotic MutS activities is required for their function in mismatch correction, and two different models for the role of ATP in MutS function have been proposed. In the translocation model, based on study of bacterial MutS, ATP binding reduces affinity of the protein for a mismatch and activates secondary DNA binding sites that are subsequently used for movement of the protein along the helix contour in a reaction dependent on nucleotide hydrolysis (Allen, D. J., Makhov, A., Grilley, M., Taylor, J., Thresher, R., Modrich, P., and Griffith, J. D. (1997) EMBO J. 16, 4467-4476). The molecular switch model, based on study of human MutSalpha, invokes mismatch recognition by the MutSalpha.ADP complex. After recruitment of downstream repair activities to the MutSalpha.mismatch complex, ATP binding results in release of MutSalpha from the heteroduplex (Gradia, S., Acharya, S., and Fishel, R.(1997) Cell 91, 995-1005). To further clarify the function of ATP binding and hydrolysis in human MutSalpha action, we evaluated the effects of ATP, ADP, and nonhydrolyzable ATP analogs on the lifetime of protein.DNA complexes. All of these nucleotides were found to increase the rate of dissociation of MutSalpha from oligonucleotide heteroduplexes. These experiments also showed that ADP is not required for mismatch recognition by MutSalpha, but that the nucleotide alters the dynamics of formation and dissociation of specific complexes. Analysis of the mechanism of ATP-promoted dissociation of MutSalpha from a 200-base pair heteroduplex demonstrated that dissociation occurs at DNA ends in a reaction dependent on ATP hydrolysis, implying that release from this molecule involves movement of the protein along the helix contour as predicted for a translocation mechanism. In order to reconcile the relatively large rate of movement of MutS homologs along the helix with their modest rate of ATP hydrolysis, we propose a novel mechanism for protein translocation along DNA that supports directional movement over long distances with minimal energy input.     

Conformational changes between the active-site and regulatory light chain of myosin as determined by luminescence resonance energy transfer: the effect of nucleotides and actin

Myosin is thought to generate movement of actin filaments via a conformational change between its light-chain domain and its catalytic domain that is driven by the binding of nucleotides and actin. To monitor this change, we have measured distances between a gizzard regulatory light chain (Cys 108) and the active site (near or at Trp 130) of skeletal myosin subfragment 1 (S1) by using luminescence resonance energy transfer and a photoaffinity ATP-lanthanide analog. The technique allows relatively long distances to be measured, and the label enables site-specific attachment at the active-site with only modest affect on myosin's enzymology. The distance between these sites is 66.8 +/- 2.3 A when the nucleotide is ADP and is unchanged on binding to actin. The distance decreases slightly with ADP-BeF3, (-1.6 +/- 0.3 A) and more significantly with ADP-AlF4 (-4.6 +/- 0.2 A). During steady-state hydrolysis of ATP, the distance is temperature-dependent, becoming shorter as temperature increases and the complex with ADP.Pi is favored over that with ATP. We conclude that the distance between the active site and the light chain varies as Acto-S1-ADP approximately S1-ADP > S1-ADP-BeF3 > S1-ADP-AlF4 approximately S1-ADP-Pi and that S1-ATP > S1-ADP-Pi. The changes in distance are consistent with a substantial rotation of the light-chain binding domain of skeletal S1 between the prepowerstroke state, simulated by S1-ADP-AlF4, and the post-powerstroke state, simulated by acto-S1-ADP.     

Tryptophan substitutions surrounding the nucleotide in catalytic sites of F1-ATPase

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.     

Characterization of ligands of a high-affinity metal-binding site in the latent chloroplast F1-ATPase by EPR spectroscopy of bound VO2+

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.     

Kinetic mechanism of monomeric non-claret disjunctional protein (Ncd) ATPase

The non-claret disjunctional protein (Ncd) is a kinesin-related microtubule motor that moves toward the negative end of microtubules. The kinetic mechanism of the monomer motor domain, residues 335-700, satisfied a simple scheme for the binding of 2'-3'-O-(N-methylanthraniloyl) (MANT) ATP, the hydrolysis step, and the binding and release of MANT ADP, where T, D, and Pi refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively, and MtN is the complex of an Ncd motor domain with a microtubule site. Rate constants k1 and k-4 are the rates of a first order step, an isomerization induced by nucleotide binding. The apparent second order rate constants for the binding steps are 1.5 x 10(6) M-1 s-1 for MANT ATP and 3.5 x 10(6) M-1 s-1 for MANT ADP (conditions, 50 mM NaCl, pH 6.9, 21 degrees C). The rate constant of the hydrolysis step (k2) was obtained from quench flow measurements of the phosphate burst phase corrected for the contribution of the rate of product release to the transient rate constant. The rate of phosphate dissociation was not measured; the value was assigned to account for a steady state rate of 3 s-1. The MtN complex is dissociated by ATP at a rate of 10 s-1 based on light scattering measurements. Dissociation constants of Ncd-nucleotide complexes from microtubules increased in the order adenosine 5'-O-(thiotriphosphate) (ATPgammaS) < ADP-AlF4 < ATP < ADP < ADP-vanadate. Comparison of the properties of Ncd with a monomeric kinesin K332 (Ma and Taylor (1997) J. Biol. Chem. 272, 717-723) showed a close similarity, except that the rate constants for the hydrolysis and ADP release steps and the steady state rate are approximately 15-20 times smaller for Ncd. There are two differences that may affect the reaction pathway. The rate of dissociation of MtN by ATP is comparable to the rate of the hydrolysis step, and N.T may dissociate in the cycle, whereas for kinesin, dissociation occurs after hydrolysis. The rate of dissociation of MtN by ADP is larger than the rate of ADP release from MtN.D, whereas for the microtubule-kinesin complex, the rate of dissociation by ADP is smaller than the rate of ADP release. The monomeric Mt.Ncd complex is not processive.     

ATP hydrolysis induces an intermediate conformational state in GroEL

The conformational properties of the molecular chaperone GroEL in the presence of ATP, its non-hydrolyzable analog 5'-adenylimidodiphosphate (AMP-PNP), and ADP have been analyzed by differential scanning calorimetry (DSC), Fourier-transform infra-red (FT-IR) and fluorescence spectroscopy. Nucleotide binding to one ring promotes a decrease in the Tm value of the GroEL thermal transition that is reversed when both rings are filled with nucleotide, indicating that the sequential occupation of the two protein rings by these nucleotides has different effects on the GroEL thermal denaturation process. In addition, ATP induces a conformational change in GroEL characterized by (a) the appearance of a reversible low temperature endotherm in the DSC profiles of the protein, and (b) an enhanced binding of the hydrophobic probe 8-anilino-naphthalene-1-sulfonate (ANS), which strictly depends on ATP hydrolysis. The similar sensitivity to K+ of the temperature range where activation of the GroEL ATPase activity, the low temperature endotherm, and the increase of the ANS fluorescence are abserved strongly indicates the existence of a conformational state of GroEL during ATP hydrolysis, different from that generated on ADP or AMP-PNP binding. To achieve this intermediate conformation, GroEL mainly modifies its tertiary and quaternary structures, leading to an increased exposure of hydrophobic surfaces, with minor rearrangements of its secondary structure.