Functions and localization of nucleotide-binding sites of CF1-ATPase using dialdehyde derivatives of ADP and ATP

The covalent binding of dialdehyde derivatives of ATP and ADP (o-ATP and o-ADP) results in inactivation of chloroplast CF1-ATPase, the degree of inactivation being increased at a rise in temperature and pH. o-ADP causes predominant inhibition of the Mg2+-dependent, while o-ATP--of both Mg2+- and Ca2+-dependent activities of CF1-ATPase. The substrates and reaction products prevent the enzyme inactivation, whereas the stimulators of the Mg2+-dependent ATPase activity enhance it. The effect of these stimulators is correlated with predominant incorporation of [3H] o-nucleotide into the beta-subunit of CF1. In the absence of the stimulators o-ADP is predominantly bound to the alpha-subunit of CF1. The binding of o-ADP and o-ATP to the beta-subunit is increased in the presence of Mg2+. A comparative analysis of the labelled nucleotides incorporation into individual subunits and the changes in the catalytic and regulatory properties of the enzyme demonstrated that the catalytic and stimulator-sensitive "regulatory" sites of the enzyme are located on the beta-subunits.     

Glutamic acid 472 and lysine 480 of the sodium pump alpha 1 subunit are essential for activity. Their conservation in pyrophosphatases suggests their involvement in recognition of ATP phosphates.

P-type ATPases such as the Na+,K+-ATPase (sodium pump) hydrolyze ATP to pump ions through biological membranes against their electrochemical gradients. The mechanisms that couple ATP hydrolysis to the vectorial ion transport are not yet understood, but unveiling structures that participate in ATP binding and in the formation of the ionophore might help to gain insight into this process. Looking at the alpha- and beta-phosphates of ATP as a pyrophosphate molecule, we found that peptides highly conserved among all soluble inorganic pyrophosphatases are also present in ion-transporting ATPases. Included therein are Glu48 and Lys56 of the Saccharomyces cerevisiae pyrophosphatase (SCE1-PPase) that are essential for the activity of this enzyme and have been shown in crystallographic analysis to interact with phosphate molecules. To test the hypothesis that equivalent amino acids are also essential for the activity of ion-transporting ATPases, Glu472 and Lys480 of the sodium pump alpha 1 subunit corresponding to Glu48 and Lys56 of SCE1-PPase were mutated to various amino acids. Mutants of the sodium pump alpha1 subunit were expressed in yeast and analyzed for their ATPase activity and their ability to bind ouabain in the presence of either ATP, Mg2+, and Na+ or phosphate and Mg2+. All four mutants investigated, Glu472Ala, Glu472Asp, Lys480Ala, and Lys480Arg, display only a fraction of the ATPase activity obtained with the wild-type enzyme. The same applies with respect to their ability to bind ouabain, where maximum ouabain binding to the mutants accounts for only about 10% of the binding obtained with the wild-type enzyme. On the basis of our results, we conclude that Glu472 and Lys480 are essential for the activity of the sodium pump. Their function is probably to arrest the alpha- and beta-phosphate groups of ATP in a proper position prior to hydrolysis of the gamma-phosphate group. The identification of these amino acids as essential components of the ATP-recognizing mechanism of the pump has resulted in a testable hypothesis for the initial interactions of the sodium pump, and possibly of other P-type ATPases, with ATP.     

Presteady-state kinetics of ATP hydrolysis by chloroplast CF1-ATPASE

The kinetics of reversible inactivation of chloroplast CF1-ATPase by Mg2+ and ADP was studied. The rate of inactivation obeys the first-order equation and is independent of ADP concentration. An analysis of the dependence of the inactivation rate on Mg2+ concentration demonstrated that the limiting step of inactivation is other than Mg2+ binding, i.e. the subsequent steps which include, in all probability, the conformational changes of the enzyme. The original Mg2+-dependent activity of CF1-ATPase is close to that observed under steady-state conditions in the presence of sulphate and methanol and exceeds the Ca2+-dependent activity approximately 6-fold. Preincubation of CF1-ATPase with Mg2+ results in inhibition of the original activity of the enzyme. This effect is not removed by addition of the ATP-regenerating system (pyruvate kinase + phosphoenol pyruvate) to the preincubation medium but is diminished by sulphite and the non-hydrolyzed analog of ATP--beta, gamma-methyladenosine-5-triphosphate. After addition of AMPPCP to the reaction mixture the initial reaction rate is decreased, while the steady-state rate is increased. It may be concluded that the Mg2+-dependent inactivation of CF1-ATPase is induced by the tightly bound ADP. The latter can be replaced by ATP, which in contrast to ADP does not form an inactive complex with the enzyme. A comparison of experimental results with literature data suggests that the mechanism of "alternating sites" proposed by Boyer et al. for ATP hydrolysis by soluble CF1-ATPase is not realized under the given experimental conditions.     

Characteristics of the combination of inhibitory Mg2+ and azide with the F1 ATPase from chloroplasts.

The interactions between ADP, Mg2+, and azide that result in the inhibition of the chloroplast F1 ATPase (CF1) have been explored further. The binding of the inhibitory Mg2+ with low Kd is shown to occur only when tightly bound ADP is present at a catalytic site. Either the tightly bound ADP forms part of the Mg(2+)-binding site or it induces conformational changes creating the high-affinity site for inhibitory Mg2+. Kinetic studies show that CF1 forms two catalytically inactive complexes with Mg2+. The first complex results from Mg2+ binding with a Kd for Mg2+ dissociation of about 10-15 microM, followed by a slow conversion to a complex with a Kd of about 4 microM. The rate-limiting step of the CF1 inactivation by Mg2+ is the initial Mg2+ binding. When medium Mg2+ is chelated with EDTA, the two complexes dissociate with half-times of about 1 and 7 min, respectively. Azide enhances the extent of Mg(2+)-dependent inactivation by increasing the affinity of the enzyme for Mg2+ 3-4 times and prevents the reactivation of both complexes of CF1 with ADP and Mg2+. This results from decreasing the rate of Mg2+ release; neither the rate of Mg2+ binding to CF1 nor the rate of isomerization of the first inactive complex to the more stable form is affected by azide. This suggests that the tight-binding site for the inhibitory azide requires prior binding of both ADP and Mg2+.     

Demonstration of cooperating alpha subunits in working (Na+ + K+)-ATPase by the use of the MgATP complex analogue cobalt tetrammine ATP

The MgATP complex analogue cobalt-tetrammine-ATP [Co(NH3)4ATP] inactivates (Na+ + K+)-ATPase at 37 degrees C slowly in the absence of univalent cations. This inactivation occurs concomitantly with incorporation of radioactivity from [alpha-32P]Co(NH3)4ATP and from [gamma-32P]Co(NH3)4ATP into the alpha subunit. The kinetics of inactivation are consistent with the formation of a dissociable complex of Co(NH3)4ATP with the enzyme (E) followed by the phosphorylation of the enzyme: (Formula: see text). The dissociation constant of the enzyme-MgATP analogue complex at 37 degrees C is Kd = 500 microM, the inactivation rate constant k2 = 0.05 min-1. ATP protects the enzyme against the inactivation by Co(NH3)4ATP due to binding at a site from which it dissociates with a Kd of 360 microM. It is concluded, therefore, that Co(NH3)4ATP binds to the low-affinity ATP binding site of the E2 conformational state. K+, Na+ and Mg2+ protect the enzyme against the inactivation by Co(NH3)4ATP. Whilst Na+ or Mg2+ decrease the inactivation rate constant k2, K+ exerts its protective effect by increasing the dissociation constant of the enzyme.Co(NH3)4ATP complex. The Co(NH3)4ATP-inactivated (Na+ + K+)-ATPase, in contrast to the non-inactivated enzyme, incorporates [3H]ouabain. This indicates that the Co(NH3)4ATP-inactivated enzyme is stabilized in the E2 conformational state. Despite the inactivation of (Na+ + K+)-ATPase by Co(NH3)4ATP from the low-affinity ATP binding site, there is no change in the capacity of the high-affinity ATP binding site (Kd = 0.9 microM) nor of its capability to phosphorylate the enzyme Na+-dependently. Since (Na+ + K+)-ATPase is phosphorylated Na+-dependently from the high-affinity ATP binding site although the catalytic cycle is arrested in the E2 conformational state by specific modification of the low-affinity ATP binding site, it is concluded that both ATP binding sites coexist at the same time in the working sodium pump. This demonstration of interacting catalytic subunits in the E1 and E2 conformational states excludes the proposal that a single catalytic subunit catalyzes (Na+ + K+)-transport.     

Active/Inactive state transitions of the chloroplast F1 ATPase are induced by a slow binding and release of Mg2+. Relationship to catalysis and control of F1 ATPases

Mg2+ is known to be a potent inhibitor of F1 ATPases from various sources. Such inhibition requires the presence of a tightly bound ADP at a catalytic site. Results with the spinach chloroplast F1 ATPase (CF1) show that the time delays of up to 1 min or more in the induction or the relief of the inhibition are best explained by a slow binding and slow release of Mg2+ rather than by slow enzyme conformational changes. CF1 is known to have multiple Mg2+ binding sites with Kd values in the micromolar range. The inhibitory Mg2+ and ADP can bind independently to CF1. When Mg2+ and ATP are added to the uninhibited enzyme, a relatively fast rate of hydrolysis attained soon after the addition is followed by a much slower steady-state rate. The inhibited steady-state rate results from a slowly attained equilibrium of binding of medium Mg2+. The Kd for the binding of the inhibitory Mg2+ is in the range of 1-8 microM, in the presence or absence of added ATP, as based on the extent of rate inhibition induced by Mg2+. Assessments from 18O exchange experiments show that the binding of Mg2+ is accompanied by a relatively rapid change to an enzyme form that is incapable of hydrolyzing MgATP. When ATP is added to the Mg2+- and ADP-inhibited enzyme, the resulting reactivation can be explained by MgATP binding to an alternate catalytic site which results in a displacement of the tightly bound ADP after a slow release of Mg2+. Both an increase in temperature (to 50 degrees C) and the presence of activating anions such as bicarbonate or sulfite reduce the extent of the Mg2+ inhibition markedly. The activating anions may bind to CF1 in place of Pi near the ADP. Whether the inhibitory Mg2+ binds at catalytic or noncatalytic nucleotide binding sites or at another location is not known. The Mg2(+)- and ADP-induced inhibition appears to be a general property of F1 ATPases, which show considerable differences in affinity for ADP, Mg2+, and Pi. These differences may reflect physiological control functions.     

Role of metal ions in coupling between chemical catalysis and conformational changes in ATP synthase

Isolated chloroplast ATP synthase (CF0F1) was used for determination of the structure-function relation by measuring the effect of divalent metal ions on the properties of ATPase. Mg2+ ions were more efficient catalysts than Ca2+ ions as indicated by Kcat/Km of 55.2 and 5.4, respectively. Other activity parameters related to binding, such as the Km of MATP and Ki of MADP, indicated a stronger binding in the presence of Mg2+ as seen from a Mg2+/Ca2+ ratio of 2.8 and 3.8, respectively. Strong binding of Ca2+ ions with a Kd of 0.03 +/- 00.6 microM-1 was detected only in the presence of ADP probably because of the positive interactive effect of CaADP as indicated in the inhibition properties. Mg2+ ions were more efficient catalysts also in other forms of the enzyme such as in the thylakoid membrane, in isolated CF0F1 and in CF1. The Mg2+/Ca2+ ratio of Kcat/Km was 5.3, 10.2 and 1.5 for the thylakoid membrane enzyme, the isolated CF0F1 and the soluble CF1 respectively. This indicated that Ca2+ ions became less efficient catalysts in the more intact and integrated enzyme while Mg2+ ions were as efficient in all forms of the enzyme. Unlike Mg2+, Ca2+ ions also did not support proton-coupled ATP synthesis and ATP driven proton pumping. It is suggested that the differences in the ligand structure of these two ions might be the reason for the differential function. An average 0.3 A shorter bond length of octahedral first coordination in Ca2+ ions caused a weaker binding of CaATP than that of MgATP. The effect of differential binding is discussed in relation to the binding of the transition state intermediate and to the rate of product release.     

Mg2+ -dependent inactivation / H+ -dependent activation equilibrium of chloroplast F1-ATPase

The catalytic part of chloroplast thylakoid ATPase, the chloroplast coupling factor CF1, is reversibly inactivated during incubation in the presence of Mg2+. The inactivation has two phases. Its fast phase occurs at basic pH of the incubation medium (k = 6 min-1), while the slow phase ( k = 0.1-0.2 min-1) depends on pH only slightly throughout the studied range (5.5-9.0). As followed from changes in the inactivation effect of magnesium ions, Mg2+ affinity for the enzyme decreases dramatically with decreasing medium pH. The pH-dependence of Mg2+ dissociation apparent constant suggests that the binding/dissociation equilibrium is determined by protonation/deprotonation of specific acid-base groups of the enzyme. The analysis of pH-dependence plots gives the equilibrium constant of magnesium dissociation (3-9 M) and the dissociation constant of the protonated groups pK 5.8-6.7). Sodium azide is known to stabilize the inactive CF1-MgADP complex; when added to the incubation medium it diminishes the Mg2+ dissociation constant and has no effect on the dissociation constant of the acid-base groups. At lower pH, Mg2+-inactivated CF1-ATPase reactivates. Octyl glucoside accelerates the reactivation, while Triton-100 affects it only slightly. The reactivation rate of membrane-bound CF1 (thylakoid ATPase) inactivated by preincubation with Mg2+ in the presence of gramicidin is a few times higher than that of isolated CF1. These results suggest that the reactivation of isolated and membrane-bound CF1-ATPase is determined by protonation of a limited number of acid-base groups buried in the enzyme molecule.     

Characterization of a phosphate binding domain on the alpha-subunit of chloroplast ATP synthase using the photoaffinity phosphate analogue 4-azido-2-nitrophenyl phosphate

The photoaffinity phosphate analogue 4-azido-2 nitrophenyl phosphate (ANPP) was shown previously (Pougeois, R., Lauquin, G. J.-M., and Vignais, P. V. (1983) Biochemistry 22, 1241-1245) to bind covalently and specifically to a single catalytic site on one of the three beta-subunits of the isolated chloroplast coupling factor 1 (CF(1)). Modification by ANPP strongly inhibited ATP hydrolysis activity. In this study, we examined labeling of membrane-bound CF(1) by ANPP by exposing thylakoid membranes to increasing concentrations of the reagent. ANPP exhibited saturable binding to two sites on CF(1), one on the beta-subunit and one on the alpha-subunit. Labeling by ANPP resulted in the complete inhibition of both ATP synthesis and ATP hydrolysis by the membrane-bound enzyme. Labeling of both sites by ANPP was reduced by more than 80% in the presence of P(i) (> or = 10 mM) and ATP (> or = 0.5 mM). ADP was less effective in competing with ANPP for binding, giving a maximum of approximately 35% inhibition at concentrations > or = 2 mM. ANPP-labeled tryptic peptides of the alpha-subunit were isolated and sequenced. The majority of the probe was contained in three peptides corresponding to residues Gln(173) to Arg(216), Gly(217) to Arg(253), and His(256) to Arg(272) of the alpha-subunit. In the mitochondrial F(1) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), all three analogous peptides are located within the nucleotide binding pocket and within close proximity to the gamma-phosphate binding site. The data indicate, however, that the azidophenyl group of bound ANPP is oriented at approximately 180 degrees in the opposite direction to the adenine binding site with reference to the phosphate binding site on the alpha-subunit. The study has confirmed that ANPP is a bona fide phosphate analogue and suggests that it specifically targets the gamma-phosphate binding site within the nucleotide binding pockets on the alpha- and beta-subunits of CF(1). The study also indicates that in the resting state of the chloroplast F(1)-F(0) complex both the alpha- and beta-subunits are structurally asymmetric.     

Stability and Activation of Glutamate Apodecarboxylase from Pig Brain

The stability and activation of glutamate apodecarboxylase was studied with three forms of the enzyme from pig brain (referred to as the alpha, beta, and gamma forms). Apoenzyme was prepared by incubating the holoenzyme with aspartate followed by chromatography on Sephadex G-25. Apoenzyme was much less stable than holoenzyme to inactivation by heat (for beta-glutamate decarboxylase (beta-GAD) at 30 degrees C, t1/2 values of apo- and holoenzyme were 17 and greater than 100 min). ATP protected holoenzyme and apoenzyme against heat inactivation. The kinetics of reactivation of apoenzyme by pyridoxal-P was consistent with a two-step mechanism comprised of a rapid, reversible association of the cofactor with apoenzyme followed by a slow conversion of the complex to active holoenzyme. The reactivation rate constant (kr) and apparent dissociation constant (KD) for the binding of pyridoxal-P to apoenzyme differed substantially among the forms (for alpha-, beta-, and gamma-GAD, kr = 0.032, 0.17, and 0.27 min-1, and KD = 0.014, 0.018, and 0.04 microM). ATP was a strong competitive inhibitor of activation (Ki = 0.45, 0.18, and 0.39 microM for alpha-, beta-, and gamma-GAD). In contrast, Pi stimulated activation at 1-5 mM but inhibited at much higher concentrations. The results suggest that ATP is important in stabilizing the apoenzyme in brain and that ATP, Pi, and other compounds regulate its activation.     

The characteristics and effect on catalysis of nucleotide binding to noncatalytic sites of chloroplast F1-ATPase.

The recent finding that the presence of ATP at non-catalytic sites of chloroplast F1-ATPase (CF1) is necessary for ATPase activity (Milgrom, Y. M., Ehler, L. L., and Boyer, P. D. (1990) J. Biol. Chem. 265,18725-18728) prompted more detailed studies of the effect of noncatalytic site nucleotides on catalysis. CF1 containing at noncatalytic sites less than one ADP or about two ATP was prepared by heat activation in the absence of Mg2+ and in the presence of ADP or ATP, respectively. After removal of medium nucleotides, the CF1 preparations were used for measurement of the time course of nucleotide binding from 10 to 100 microM concentrations of 3H-labeled ADP, ATP, or GTP. The presence of Mg2+ strongly promotes the tight binding of ADP and ATP at noncatalytic sites. For example, the ADP-heat-activated enzyme in presence of 1 mM Mg2+ binds ADP with a rate constant of 0.5 x 10(6) M-1 min-1 to give an enzyme with two ADP at noncatalytic sites with a Kd of about 0.1 microM. Upon exposure to Mg2+ and ATP the vacant noncatalytic site binds an ATP rapidly and, as an ADP slowly dissociates, a second ATP binds. The binding correlates with an increase in the ATPase activity. In contrast the tight binding of [3H]GTP to noncatalytic sites gives an enzyme with no ATPase activity. The three noncatalytic sites differ in their binding properties. The noncatalytic site that remains vacant after the ADP-heat-activated CF1 is exposed to Mg2+ and ADP and that can bind ATP rapidly is designated as site A; the site that fills with ATP as ADP dissociates when this enzyme is exposed to Mg2+ and ATP is called site B, and the site to which ADP remains bound is called site C. Procedures are given for attaining CF1 with ADP at sites B and C, with GTP at sites A and/or B, and with ATP at sites A, B, and/or C, and catalytic activities of such preparations are measured. For example, little or no ATPase activity is found unless ATP is at site A, but ADP can remain at site C with no effect on ATPase. Maximal GTPase activity requires ATP at site A but about one-fifth of maximal GTPase is attained when GTP is at sites A and B and ATP at site C. Noncatalytic site occupancy can thus have profound effects on the ATPase and GTPase activities of CF1.     

Magnesium ion-induced changes in the binding mode of adenylates to chloroplast coupling factor 1.

The effect of Mg2+ on the binding of adenylates to isolated chloroplast coupling factor 1 (CF1) was studied using CD spectrometry and ultrafiltration. At adenylate concentrations smaller than 100 muM, one mole of CF1 binds three moles of ATP (or ADP) regardless of the presence of Mg2+. In the presence of Mg2+, the first two ATP's bind to CF1 independently with the same binding constant of 2.5 X 10(-1) muM-1, then the third ATP binds with a much higher affinity of 10 muM-1. In the absence of Mg2+, the first ATP binds to CF1 with a binding constant of 2.5 X 10(-1) muM-1 then the other two ATP's bind less easily with the same binding constant of 4.0 X 10(-2) muM-1. The binding mode of ADP to CF1 is quite similar to that of ATP. In the presence of Mg2+, the binding constants of the first two ADP's are both 7.6 X 10(-2) muM-1, that of the third ADP being 4.0 muM-1. In the absence of Mg2+, the binding constant of the first ADP is 7.6 X 10(-2) muM-1, the constants of the other two ADP's both being 4.0 X 10(-2) muM-1. AMP caused a negligible change in CD.     

Characterization of three-subunit chloroplast coupling factor

The delta- and epsilon-polypeptides were removed from chloroplast coupling factor 1 (CF1). The resulting enzyme, CF1(-delta, epsilon), is a stable active ATPase containing only alpha-, beta-, and gamma-polypeptides. The dependence of the steady-state kinetics of ATP hydrolysis catalyzed by CF1(-delta, epsilon) on the concentrations of ATP and ADP was found to be essentially the same as by activated CF1. Nucleotide binding studies with CF1(-delta, epsilon) revealed three binding sites: a nondissociable ADP site (site 1), a tight MgATP binding site (site 2), and a site that binds ADP and ATP with a dissociation constant in the micromolar range (site 3). Similar results have been obtained with CF1. For both CF1 and CF1(-delta, epsilon), the binding of MgATP at site 2 is tight only in the presence of Mg2+. Fluorescence resonance energy transfer was used to map distances between the gamma-sulfhydryl ("dark" site) and gamma-disulfide and between the gamma-sulfhydryl and the three nucleotide sites. These distances are within 5% of the corresponding distances on CF1. These results indicate that removal of the delta- and epsilon-polypeptides from CF1 does not cause significant changes in the structure, kinetics, and nucleotide binding sites of the enzyme.     

The separation of subunits of phenylalanyl-tRNA-synthetase from Escherichia coli MRE-600 by means of affinity chromatography in dissociation conditions

The method of affinity chromatography on sepharose with immobilized tRNA in the presence of urea was developed for separating the subunits of phenylalanyl-tRNA synthetase from E. coli MRE-600 (subunit structure alpha 2 beta 2). Specific binding of large beta-subunits of the enzyme on immobilized tRNA testifies the localization of the tRNA-binding center on the beta-subunit of phenylalanyl-tRNA synthetase. Separately alpha- and beta-subunits of the enzyme exhibit no catalytic activity. Incubation of the mixture of alpha- and beta-subunits in conditions leading to reassociation of the oligomeric structure results in restoration of catalytic activity of the enzyme. In the presence of urea resin with immobilised analogs of ATP binds alpha- and beta-subunits of the enzyme. This testifies the presence of nucleotide-binding sites on both subunits. The possibility of using the affinity chromatography method to separate non-identical subunits of different enzymes is discussed.     

The effects of octylglucoside on the interactions of chloroplast coupling factor 1 (CF1) with adenine nucleotides

The effects of octylglucoside (OcGlc) micelles, which stimulate a Mg-specific ATPase activity in chloroplast coupling factor 1 [Pick, U. and Bassilian, S. (1982) Biochemistry, 21, 6144-6152], on the interactions of the enzyme with adenine nucleotides have been studied. 1. OcGlc specifically accelerates the binding and the release of ADP but not of ATP or adenosine 5'[beta, gamma-imido]triphosphate (AdoPP[NH]P) from the tight-sites. The binding affinity for ADP and for ATP is only slightly decreased (twofold) by the detergent. ATP competitively inhibits the binding of ADP and vice versa in the presence or absence of OcGlc. 2.OcGlc-induced inactivation of CF1-ATPase is correlated with the release of bound nucleotides. In the absence of medium nucleotides ADP X CF1 is rapidly inactivated while ATP X CF1 and AdoPP[NH]P X CF1 are slowly inactivated by OcGlc in parallel with the release of bound nucleotide. In contrast, low concentrations of either ATP or ADP in the medium effectively protect against OcGlc inactivation while AdoPP[NH]P, whose binding to CF1 is inhibited by OcGlc, is ineffective even at millimolar concentrations. The results suggest that the occupancy of the tight-sites protects the enzyme against OcGlc-induced inactivation. 3. Mg ions specifically inhibit the release of bound ADP and the OcGlc-induced inactivation of CF1. High concentrations of medium ATP and ADP (K50 = 100 microM) also inhibit the OcGlc-induced release of bound nucleotides in an EDTA medium. In contrast, in the absence of OcGlc, medium ADP and ATP accelerate the release of bound adenine nucleotides. 4. Mg-ATP in the presence of OcGlc stimulates the release of bound ADP from CF1. Bound ATP is neither released nor hydrolyzed at the tight-sites under these conditions where medium ATP is rapidly hydrolyzed. Mg-ADP stimulates the release of bound ADP only in the presence of inorganic phosphate or of phosphate analogs, e.g. arsenate, pyrophosphate or selenate. 5. It is suggested that: (a) ATP and ADP bind to the same tight-sites, but OcGlc activation specifically accelerates the exchange of bound ADP at the site. (b) CF1 contains low affinity adenine nucleotide binding sites which may be the catalytical sites and which influence the tight-sites by cooperative interactions. (c) Mg-ATP in the presence of OcGlc induces a conformational change at the catalytical site which accelerates the release of ADP from the tight-site. The implications of these results to the role of adenine nucleotides in the regulation and mechanism of ATP hydrolysis by CF1 are discussed.     

Photolabelling with 8-azido-adenine nucleotides of adenine nucleotide-binding sites in isolated spinach chloroplast ATPase (CF1)

1. Photolabelling of chloroplast ATPase (CF1) with either 8-azido-ATP or 8-azido-ADP leads to inactivation of the ATPase activity. ATP and ADP protect against the inactivation, whereas AMP dose not. 2. Ca2+ has little if any effect on the degree of inactivation by photolabelling with 8-azido-ADP, but, at the same degree of inactivation, twice as much label is bound in the presence of Ca2+ as in its absence. 3. The degree of inactivation of ATPase and the amount of bound photolabel are independent of the extent of pre-activation of the CF1. 4. Upon extrapolation to complete inactivation, 2 mol label, either 8-azido-ATP or 8-azido-ADP can be bound. 5. In all cases the label is bound specifically to the alpha and beta subunits in almost equal amounts. The location of the bound label is not affected by addition of Ca2+, ATP or ADP.     

Affinity labeling of the ATP-binding site of type II calmodulin-dependent protein kinase by 5'-p-fluorosulfonylbenzoyl adenosine

Modification of the type II calmodulin-dependent protein kinase by 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) resulted in a time-dependent inactivation of the enzyme. The reaction followed pseudo-first-order kinetics and showed a nonlinear dependence on reagent concentration. The rate of inactivation was sensitive to Mg2+- and calmodulin-induced conformational changes on the enzyme. However, the enhancing effects of these ligands were not additive; indeed, the kinetic parameters of the Mg2+-stimulated inactivation reaction with FSBA (Kinact = 2.4 mM; kappa max = 0.12 min-1) were almost unaffected by the simultaneous addition of calmodulin (Kinact = 1.5 mM; kappa max = 0.086 min-1). Protection from inactivation by FSBA was provided by Mg2+-ADP which is consistent with modification of the catalytic site. An analysis of the protective effect of Mg2+-ADP in the absence (Kd = 590 microM) and presence (Kd = 68 microM) of calmodulin demonstrated that binding of the modulator protein to the enzyme increases the affinity of the protein kinase for nucleotides. Modification by FSBA resulted in labeling of both Tyr and Lys residues but only labeling of Lys was decreased by Mg2+-ADP which is consistent with the hypothesis that a conserved Lys residue is important in nucleotide binding to the protein kinase. However, the kinetic results of the inactivation reaction suggest that this Lys is not involved in mediating the calmodulin-promoted increase in the affinity of the enzyme for Mg2+-nucleotide complexes.     

Limited proteolysis reveals low-affinity binding of N-acetyl-L-glutamate to rat-liver carbamoyl-phosphate synthetase (ammonia)

Carbamoyl-phosphate synthetase was inactivated by elastase with first-order kinetics, and N-acetyl-L-glutamate speeded inactivation. From the dependence of the t1/2 value for inactivation on the concentration of acetylglutamate we estimate a Kd value for binding of the activator of 0.365 mM, which is approximately 600 times greater than in the presence of ATP, HCO3-, K+ and Mg2+. K+ and Mg2+ are not required for binding with low affinity, and in the absence of ATP they do not appear to increase the affinity for acetylglutamate. In the presence of acetylglutamate, mixtures of ATP, K+ and Mg2+ protect the enzyme from inactivation. ADP or AdoPP[NH]P partly replaced ATP in protecting the enzyme and thus binding of the nucleotide without further reaction is enough for protection. Two partial activities of the enzyme were inactivated by elastase to the same extent as the overall reaction, and thus elastase affects some property of the enzyme which is essential for catalysis. With other proteinases tested, inactivation was also accelerated by acetylglutamate and was slowed by mixtures of ATP, K+, Mg2+ and acetylglutamate, suggesting that changes in the accessibility of susceptible bonds are responsible for the changes in the degree of inactivation. It is concluded that elastase attacks at or close to the binding sites for ATP, and that exposure of the binding site for the ATP molecule that yields Pi (ATPA) upon binding of acetylglutamate causes the acceleration of the proteolytic inactivation.     

Lys691 and Asp714 of the Na+/K+-ATPase alpha subunit are essential for phosphorylation, dephosphorylation, and enzyme turnover

P-type ATPases such as the sodium pump appear to be members of a superfamily of hydrolases structurally typified by the L-2-haloacid dehalogenases. In the dehalogenase L-DEX-ps, Lys151 serves to stabilize the excess negative charge in the substrate/reaction intermediates and Asp180 coordinates a water molecule that is directly involved in ester intermediate hydrolysis. To investigate the importance of the corresponding Lys691 and Asp714 of the sodium pump alpha subunit, sodium pump mutants were expressed in yeast and analyzed for their properties. Lys691Ala, Lys691Asp, Asp714Ala, and Asp714Arg mutants were inactive, not only with respect to ATPase activity but also to interaction with the highly sodium pump-specific inhibitors ouabain or palytoxin (PTX). In contrast, conservative mutants Lys691Arg and Asp714Glu retained some of the partial activities of the wild-type enzyme, although they completely failed to display any ATPase activity. Yeast cells expressing Lys691Arg and Asp714Glu mutants are sensitive to the sodium pump-specific inhibitor PTX and lose intracellular K+. Their sensitivity to PTX, with EC50 values of 118 +/- 24 and 76.5 +/- 3.6 nM, respectively, was clearly reduced by almost 7- or 4-fold below that of the native sodium pump (17.8 +/- 2.7 nM). Ouabain was recognized under these conditions with low affinity by the mutants and inhibited the PTX-induced K+ efflux from the yeast cells. The EC50 for the ouabain effect was 183 +/- 20 microM for Lys691Arg and 2.3 +/- 0.08 mM for the Asp714Glu mutant. The corresponding value obtained with cells expressing the native sodium pump was 69 +/- 18 microM. In the presence of Pi and Mg2+, none of the mutant sodium pumps were able to bind ouabain. When Mg2+ was omitted, however, both Lys691Asp and Asp714Glu mutants displayed ouabain binding that was reduced by Mg2+ with an EC50 of 0.76 +/- 0.11 and 2.3 +/- 0.2 mM, respectively. In the absence of Mg2+, ouabain binding was also reduced by K+. The EC50 values were 1.33 +/- 0.23 mM for the wild-type enzyme, 0.93 +/- 0.2 mM for the Lys691Arg mutant, and 1.02 +/- 0.24 mM for the Asp714Glu enzyme. None of the neutral or nonconservative mutants displayed any ouabain-sensitive ATPase activity. Ouabain-sensitive phosphatase activity, however, was present in membranes containing either the wild-type (1105 +/- 100 micromol of p-nitrophenol phosphate hydrolyzed min(-1) mg of protein(-1)) or the Asp714Glu mutant (575 +/- 75 micromol min(-1) mg(-1)) sodium pump. Some phosphatase activity was also associated with the Lys691Arg mutant (195 +/- 63 micromol min(-1) mg(-1)). The results are consistent with Lys691 and Asp714 being essential for the phosphorylation/dephosphorylation process that allows the sodium pump to accomplish the catalytic cycle.     

Characterization of nucleotide binding sites of the isolated H(+)-ATPase from spinach chloroplasts, CF(0)F(1)

Soluble purified CF(0)F(1) from chloroplasts was either oxidized or reduced and then incubated with [alpha-(32)P]ATP in the presence or in the absence of Mg(2+). Depending on the conditions of incubation, the enzyme showed different tight-nucleotide binding sites. In the presence of EDTA, two sites bind [alpha-(32)P]ATP from the reaction medium at different rates. Both sites promote ATP hydrolysis, since equimolar amounts of [alpha-(32)P]ATP and [alpha-(32)P]ADP are bound to the enzyme. In the presence of Mg(2+), only one site appears during the first hour of incubation, with characteristics similar to those described in the absence of Mg(2+). However, after this time a third site appears also permitting binding of ATP from the reaction medium, but in this case the bound ATP is not hydrolyzed. Covalent derivatization by 2-azido-[alpha-(32)P]ATP was used to distinguish between catalytic and noncatalytic sites. In the presence of Mg(2+), there are at least three distinct nucleotide binding sites that bind nucleotide tightly from the reaction medium: two of them are catalytic and one is noncatalytic.