Sodium binding to and protonation of ATP: a multinuclear magnetic double resonance study at 8.46 tesla

We show that the interaction of ATP with Na+ and H+, whether binding or dissociation, gives rise to exchange broadened 31P-NMR spectra at 8.4 T, pH 6.7 and 310 K. We interpret the effect as being due to a two-step conversion between two NMR-differentiated ATP pools. A quantitative analysis yields all involved equilibrium constants and some of the dynamic parameters. Our results help to understand previous studies of magnesium binding to ATP and the appearance of high-field in vivo 31P-NMR spectra.     

A study on the exchange rate of magnesium with ATP

The exchange process of Mg²⁺ with ATP was found to be, in many cases, dominated by Mg²⁺ exchange between ATP and ATP-Mg (a bimolecular reaction) rather than the Mg²⁺ off-process from ATP-Mg to solutino (a unimolecular reaction). The Mg²⁺ off-rate from ATP-Mg and the rate constant of the bimolecular reaction were determined at 10 and 25°C at pH. 7.3, using ³¹P-NMR at 145.7 MHz. At this resonance frequency intermediate to slow exchange phenomena with respect to the NMR time scale of 2.5·10³ s^?1 were observed in ATP resonances. Various implications of these results to studies of biological systems have been pointed out.     

Effect of the weakly acidic uncoupler 2,4-dinitrophenol and dimethyl sulfoxide on the coordination of Mg2+ with ATP. Possible mechanism of activation of the isolated F1-ATPase by 2,4-dinitrophenol

The exchange rate constants between Mg2(+)-free and Mg2(+)-bound ATP were determined under various conditions by line shape analysis of the 31P-NMR spectrum based on the exchange reaction, and the thermodynamic parameters of this exchange reaction were determined from the temperature dependence of its rate constants. Analysis of the activation enthalpy change delta H showed that Mg2+ is coordinated with the beta- and gamma-phosphoryl groups of ATP asymmetrically, being in closer proximity to the beta-phosphoryl group. The weakly acidic uncoupler 2,4-dinitrophenol increased this asymmetric coordination of Mg2+, and this effect was enhanced by the further addition of dimethyl sulfoxide. The hydrolysis of ATP in aqueous solution correlated well with the degree of asymmetry of Mg2+ coordination. Thus, this asymmetric coordination specifically weakens the O-P gamma bond at which specific cleavage of ATP catalyzed by most ATPases takes place in the presence of Mg2+. In this paper, the mechanism of activation of isolated ATPase (F1-ATPase) by 2,4-dinitrophenol, and that of ATP synthesis by isolated F1-ATPase in the presence of dimethyl sulfoxide are considered on the basis of these results. The essential role of the OH group of Ser-174 of the beta-subunit of F1-ATPase in ATP hydrolysis is also discussed.     

ATP/ADP exchange activity of gastric (H+ +K+)-ATPase

The ATP/ADP exchange is shown to be a partial reaction of the (H+ +K+)-ATPase by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to P1, P5-di(adenosine-5') pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg2+ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration (K0.5=116 microM) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg2+ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K+ as a function of pH and Mg2+.     

trans-1,2-Diaminocyclohexane-N,N,N',N'-tetraacetic acid is superior to ethylenediamine-N,N,N',N'-tetraacetic acid for sequestering Mg2+ in 31P NMR experiments involving ATP spectra at neutral and acidic pH

The use of ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) to sequester Mg2+ from samples containing ATP at acidic or neutral pH prior to 31P NMR spectroscopic analysis leads to significant broadening of the gamma- and beta-phosphorus resonances of ATP as compared to ATP alone. It was found that the use of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) reduces the broadening of the ATP resonances. At pH 7.0, 30 mM EDTA in the presence of 5 mM ATP and 7 mM Mg2+ leads to a threefold increase in the peak width of the gamma phosphorus of ATP as compared to 5 mM ATP alone. When 30 mM CDTA is used in the place of EDTA, the peak width decreased to about 80% of the peak width of ATP alone. When the experiment is repeated at pH 8.5, both EDTA and CDTA lead to narrow peak widths with no significant difference between the two spectra. At pH 6.0, the use of EDTA leads to a spectrum that is very noisy, with a 10-fold increase in the peak width as compared to ATP in the absence of Mg2+ at this pH, whereas the increase with CDTA is only 50%. These results do not reflect the difference in chelating strength between EDTA and CDTA: The free Mg2+ concentration in the presence of each chelator, as calculated by the computer program given in the Appendix, was nearly equal at each pH. The results, however, reflect a difference in the lability of the metal-ligand bond between EDTA and CDTA.     

Studies on the mechanism of decreased NMR-measured free magnesium in stored erythrocytes

31P-NMR spectra have been recorded on erythrocytes stored at 4 degrees C in various preservation media. Storage was always associated with an upfield shift of the inorganic phosphate (Pi) resonance and a pronounced upfield shift of the ATP beta resonance, indicating decreased intracellular pH (pHi) and decreased intracellular free magnesium ([Mg2+]i). The decreased [Mg2+]i occurred in preservation media not containing citrate and even in media supplemented with Mg2+. It could not be attributed to the changes in pHi, Na+, K+, lactate, Pi or 2,3-diphosphoglycerate, that occur with storage. The decrease in [Mg2+]i was largely reversed when stored cells were incubated for 1 h at 37 degrees C in fresh plasma. Stored cells were found to contain significant amounts of inorganic pyrophosphate, up to about 200 mumol per liter cell water. Being a tight binder of Mg2+, pyrophosphate could account for some of the observed decrease in [Mg2+]i. Additional mechanisms may involve precipitation of some other Mg2+ complex during cold storage or enhancement of Mg2+ binding to membrane components.     

31P-NMR measurements of ATP, ADP, 2,3-diphosphoglycerate and Mg2+ in human erythrocytes

Absolute 31P-NMR measurements of ATP, ADP and 2,3-diphosphoglycerate (2,3-DPG) in oxygenated and partly deoxygenated human erythrocytes, compared to measurements by standard assays after acid extraction, show that ATP is only 65% NMR visible, ADP measured by NMR is unexpectedly 400% higher than the enzymatic measurement and 2,3-DPG is fully NMR visible, regardless of the degree of oxygenation. These results show that binding to hemoglobin is unlikely to cause the decreased visibility of ATP in human erythrocytes as deoxyhemoglobin binds the phosphorylated metabolites more tightly than oxyhemoglobin. The high ADP visibility is unexplained. The levels of free Mg2+ [( Mg2+]free) in human erythrocytes are 225 mumol/l at an oxygen saturation of 98.6% and instead of the expected increase, the level decreased to 196 mumol/l at an oxygen saturation of 38.1% based on the separation between the alpha- and beta-ATP peaks. [Mg2+]free in the erythrocytes decreased to 104 mumol/l at a high 2,3-DPG concentration of 25.4 mmol/l red blood cells (RBC) and a normal ATP concentration of 2.05 mmol/l RBC. By increasing the ATP concentration to 3.57 mmol/l RBC, and with a high 2,3-DPG concentration of 24.7 mmol/l RBC, the 31P-NMR measured [Mg2+]free decreased to 61 mumol/l. These results indicate, that the 31P-NMR determined [Mg2+]free in human erythrocytes, based solely on the separation of the alpha- and beta-ATP peaks, does not give a true measure of intracellular free Mg2+ changes with different oxygen saturation levels. Furthermore the measurement is influenced by the concentration of the Mg2+ binding metabolites ATP and 2,3-DPG. Failure to take these factors into account when interpreting 31P-NMR data from human erythrocytes may explain some discrepancies in the literature regarding [Mg2+]free.     

Differences in G-actin containing bound ATP or ADP: the Mg2+-induced conformational change requires ATP

The role of adenosine 5'-triphosphate (ATP) in the Mg2+-induced conformational change of rabbit skeletal muscle G-actin has been investigated by comparing actin containing bound ADP with actin containing bound ATP. As previously described [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886], N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled G-actin containing ATP undergoes a time-dependent Mg2+-induced fluorescence change that reflects a conformational change in the actin. Addition of Mg2+ to labeled G-actin containing ADP gives no fluorescence change, suggesting that the conformational change does not occur. The fluorescence change can be restored on the addition of ATP. Examination of the time courses of these experiments suggests that ATP must replace ADP prior to the Mg2+-induced change. The Mg2+-induced polymerization of actin containing ADP is extraordinarily slow compared to that of actin containing ATP. The lack of the Mg2+-induced conformational change, which is an essential step in the Mg2+-induced polymerization, is probably the cause for the very slow polymerization of actin containing ADP. On the other hand, at 20 degrees C, at pH 8, and in 2 mM Mg2+, the elongation rate from the slow growing end of an actin filament, measured by using the protein brevin to block growth at the fast growing end, is only 4 times slower for actin containing ADP than for actin containing ATP.     

Phosphoenzymes formed from Mg.ATP and Ca.ATP during pre-steady state kinetics of sarcoplasmic reticulum ATPase

We have investigated here the pre-steady state kinetics of sarcoplasmic reticulum ATPase incubated under conditions where significant amounts of Mg.ATP and Ca.ATP coexist, both of them being substrates for the ATPase. We confirmed that these two substrates are independently hydrolyzed by the ATPase, which thus apparently catalyzes Pi production by two simultaneous and separate pathways. External calcium (or the Ca2+/Mg2+ ratio) determines the extent to which Ca2+ or Mg2+ is bound at the phosphorylation site, while internal calcium controls the rate of processing of both the slow, calcium-containing and the fast, magnesium-containing phosphoenzyme. Time-dependent binding of calcium at the catalytic site is correlated with the observed burst of Pi liberation, which therefore results from reequilibration during pre-steady state of magnesium- and calcium-containing phosphoenzyme pools. Independently of direct exchange of metal at the catalytic site, ADP produced by the hydrolysis reaction contributes to reequilibration of these pools through reversal of phosphorylation by the ATP-ADP exchange pathway.     

Interaction of potassium and magnesium with the high affinity calcium-binding sites of the sarcoplasmic reticulum calcium-ATPase.

The sarcoplasmic reticulum Ca2(+)-ATPase of skeletal muscle has two high affinity calcium sites, one of fast access ("f" site) and one of slow access ("s" site). In addition to Ca2+ these sites are able to interact with other cations like Mg2+ or K+. We have studied with a stopped-flow method the modifications produced by Mg2+ and K+ on the kinetics of the intrinsic fluorescence changes produced by Ca2+ binding to and dissociation from the Ca2(+)-ATPase of sarcoplasmic reticulum. The presence of Mg2+ ions (K1/2 = 0.5 mM at pH 7.2) leads to the appearance of a rapid phase in the Ca2+ binding, which represents half of the signal amplitude at optimal Mg2+. The presence of K+ greatly accelerates both the Ca2+ binding and the Ca2+ dissociation reactions, giving, respectively, a 4- and 8-fold increase of the rate constant of the induced fluorescence change. K+ ions also increase the rate of the 45Ca/40Ca exchange reaction at the s site measured by rapid filtration. These results lead us to build up a model for the Ca2(+)-binding mechanism of the sarcoplasmic reticulum Ca2(+)-ATPase in which Mg2+ and K+ participate at particular steps of the reaction. Moreover, we propose that, in the absence of Ca2+, this enzyme may be the pathway for monovalent ion fluxes across the sarcoplasmic reticulum membrane.     

Isolation and characterization of a highly phosphorylated troponin from bovine heart

A modified procedure for isolation of troponin from bovine heart is described, which results in a stable and highly phosphorylated protein. 31P-NMR spectra show up to four phosphoserine signals indicating that at least four serine residues of cardiac troponin are phosphorylated in the intact organ. The hydrodynamic parameters of phosphotroponin are almost identical to those previously published. Characteristically cardiac troponin shows a strong tendency to associate that is dependent on protein concentration. Mg2+ may specifically induce an aggregation, which can be observed during sedimentation. This phenomenon seems to be analogous to the Mg2+-induced dimerization of cardiac troponin C [Jaquet, K. and Heilmeyer, L. M. G., Jr (1987) Biochem. Biophys. Res. Commun. 145, 1390-1396]. Upon Mg2+ saturation a shift of one of the four 31P-NMR signals is observed. The affinity of troponin to Ca2+ is reduced when the protein concentration is enhanced only in the presence of Mg2+. This effect of Mg2+ suggests a model for the regulation of the Ca2+-binding affinity of cardiac troponin.     

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.     

Activation of partial reactions of the Ca2+-ATPase from human red cells by Mg2+ and ATP.

(1) At ATP concentrations up to 30 micrometer addition of 0.5 mM MgCl2 in the reaction mixture increases both the rate of formation and the steady-state level of the phosphoenzyme of the Ca2+-ATPase from human red cell membranes. Under these conditions Mg2+ has no effect on the rate of dephosphorylation, which remains slow. (2) In the presence of Mg2+ the rate of dephosphorylation is increased 5 to 10 times by high (1 mM) concentrations of ATP. (3) Provided Mg2+ has reacted with the phosphoenzyme, acceleration of dephosphorylation by ATP takes place in the absence of Mg2+. This suggests that the role of Mg2+ on dephosphorylation is to convert the phosphoenzyme into a form that, after combination with ATP, reacts rapidly with water. (4) The results are consistent with the idea that combination of ATP at a non-catalytic site is needed for rapid dephosphorylation of the Ca2+-ATPase.     

31P NMR of Mg-ATP in dilute solutions: complexation and exchange.

1. Monovalent-cation [(CH3)4N+, K(I), Na(I)] ATP, 1 mM in nucleotide, in aqueous solutions at pH 7.2, 24 degrees C, generates 2 different 31P NMR spectra, depending upon the salt content of the solution. At salt concentrations below 10 mM, the 31P NMR signals are chemically-shifted upfield (Na salt: alpha, -11.44 delta; beta, -22.91 delta; gamma, -8.36 delta) and the beta- and gamma-groups are broadened (at half-height: alpha, 3.5 Hz; beta, 9.6 Hz; gamma, 69 Hz). Above 10 mM salt, the signals are shifted downfield and are narrow (Na salt: alpha, -11.09 delta, 1.9 Hz; beta, -21.75 delta, 3.3 Hz; gamma, -6.30 delta, 3.9 Hz). 2. The Na-Mg-ATP complex, corresponding to the composition Na6Mg1ATP2, yields a single set of 31P resonances at concentrations of nucleotide of 100 mM, that upon dilution to 0.2 mM, resolve into 2 sets of ATP resonances characterized by low-field and high-field beta- and gamma-group resonance pairs. This set of ATP resonances, in contrast to the resonance set at 100 mM ATP, are broad (100 mM in ATP: alpha, -10.7 delta, 3.7 Hz; beta, -20.1 delta, 15 Hz; gamma, -5.7 delta, 7.3 Hz. 0.2 mM in ATP: alpha, -10.7 delta, 47 Hz; beta, -18.8 and -21.6 delta, 316 and 274 Hz; gamma, -5.5 and -8.7 delta, 460 and 374 Hz). 3. This new data, in combination with data derived from a survey of metal-ion-ATP studies, are interpreted in terms of ATP dimers, incorporating 2 molecules of ATP and 2 metal cations, that exist in water under the physiological conditions of neutral pH, high salt content [135 mM K(I)] and ATP concentrations in the range of 3 mM. 4. A compilation of 31P in vivo and ex vivo data compared to a reference Mg-ATP chemical shift vs Mg/ATP ratio plot indicates that ATP is not fully Mg-saturated in living systems and that 41% exists as the Mg(ATP)2 complex.     

Phosphoryl Group Exchange between ATP and ADP Catalyzed by H+-ATPase from Oat Roots

ATP-ADP exchange was estimated in the presence of plasma membrane H+-ATPase of oat (Avena sativa) roots partially purified with Triton X-100 by measuring [14C]ATP formation from [14C]ADP. Most studies were done at 0[deg]C. At pH 6.0 the exchange showed: (a) Mg2+ requirement with a biphasic response giving maximal activity at 152 [mu]M and (b) insensitivity to ionic strength, [Na+], and [K+]. ATP and ADP dependence were analyzed with a model in which nucleotide-enzyme interactions are at rapid-random equilibrium, whereas E1ATP [left right arrow] E1P-ADP transitions occur in steady state. The results indicated competition between ADP and ATP for the catalytic site, whereas ATP interaction with the ADP site was extremely weak. At 0[deg]C the exchange showed a 3-fold pH increase, from pH 5.5 to 9.0. At an alkaline pH the reaction was not affected by sodium azide and carbonyl cyanide p-trifluometoxyphenyl-hydrazone, had a biphasic response to Mg2+ (maximal at 513 [mu]m), and was insensitive to ionic strength. At 20[deg]C ATP-ADP exchange was pH insensitive. At both temperatures ATP hydrolysis displayed a bell-shaped response, with a maximum around pH 6.0 to 6.5. Because no adenylate kinase activity was detected under any condition, these results demonstrate the existence of an ATP-ADP exchange reaction catalyzed by the plant H+-ATPase.     

Catalytic properties of Escherichia coli polyphosphate kinase: an enzyme for ATP regeneration.

Catalytic properties of Escherichia coli polyphosphate kinase (EC 2.7.4.1), a promising enzyme for use in ATP regeneration (Hoffman, et al., 1988, Biotechnol. Appl. Biochem. 10, 107-117), are reported here. E. coli polyphosphate kinase (PPK) is broadly active in the pH range 5.5 to 8.5, having an optimal Vmax at pH 7.2. The Km values for the substrates, ADP and polyphosphate (Pn), change little in the same pH range. The optimal concentration range for the Mg2+ activator is 1-20 mM, with an activity maximum at 10 mM Mg2+. In addition to Mg2+, Mn2+ and Co2+ can serve as activators of E. coli PPK, whereas Zn2+ and Cu2+ are highly inhibitory. E. coli PPK is most active with Pn substrates of chain length greater than 132 phosphoryl units. The enzyme activity decreases with decreasing Pn chain length and approaches zero (less than 1%) at a chain length less than or equal to 5. Equilibrium yields of ATP of greater than 85% are readily attained at substrate concentrations below 1 mM. An operational equilibrium constant for the PPK reaction, defined as [ATP]/[ADP][Pn], was determined to be 7.5 (+/- 3.4) x 10(5) M-1. The data presented here serve as a base of information from which assessments of the suitability of E. coli PPK for specific ATP regeneration applications can be made.     

Interactions between K+ and ATP binding to the (Na+ + K+)-dependent ATPase.

K+ appears to decrease the affinity of the (Na+ + K+)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) for its substrate, Mg2+ - ATP, and Mg2+ - ATP, in turn, appears to decrease the affinity of the enzyme for K+. These antagonisms have been investigated in terms of a quantitative model defining the magnitude of the effects as well as identifying the class of K+ sites on the enzyme involved. K+ increased the apparent Km for Mg2+ - ATP, an effect that was antagonized competitively by Na+. The data can be fitted to a model in which Mg2+ - ATP binding is prevented by occupancy of alpha-sites on the enzyme by K+ (i.e. sites of moderate affinity for K+ accessible on the "free" non-phosphorylated enzyme, in situ on the external membrane surface). By contrast, occupancy of these alpha-sites by Na+ has no effect on Mg2+ - ATP binding to the enzyme. On the other hand, Mg2+ - ATP decreased the apparent affinity of the enzyme for K+ at the alpha-sites, in terms of (i) the KD for K+ measured by K+-accelerated inactivation of the enzyme by F-, and (ii) the concentration of K+ for half-maximal activation of the K+-dependent phosphatase reaction (which reflects the terminal hydrolytic steps of the overall ATPase reaction). These data fit the same quantitative model. Although this formulation does not support schemes in which ATP binding effects the release of transported K+ from discharge sites, it is consistent with observations that K+ can inhibit the enzyme at low substrate concentrations, and that Li+, which has poor efficacy when occupying these alpha-sites, can stimulate enzymatic activity at high K+ concentrations by displacing the inhibitory K+.     

Regulation of the synthesis and hydrolysis of ATP by mitochondrial ATPase. Role of Mg2+

ATP hydrolysis, the exchange of inorganic phosphate with ATP, and ATP synthesis have been studied as a function of Mg2+ concentration in bovine heart submitochondrial particles. The rate of exchange is low at concentrations of Mg2+ below 3 mM, at higher concentrations, the exchange is several times higher. ATP hydrolysis shows a different pattern with respect to the concentration of Mg2+. The ratio of ATP hydrolyzed to ATP exchanged is above 20 at Mg2+ concentrations below 3 mM and about 5 at high Mg2+ concentrations; ADP induces a further drop of the ratio (2-3). By assays of the sensitivity of the hydrolytic reaction to organic solvents (dimethyl sulfoxide), it has been possible to determine the rate-limiting step of ATP hydrolysis. At 3 mM Mg2+, the rate-limiting step is the release of ADP in the soluble, but not in the particulate enzyme. However at higher Mg2+ concentrations, the rate-limiting step in the particulate enzyme is also ADP release. Therefore, the decrease in the ratio of ATP hydrolysis to inorganic phosphate incorporated into ATP coincides with a change in the kinetics of the enzyme, i.e. when the terminal step of ATP hydrolysis becomes rate-limiting, the inorganic phosphate-ATP exchange increases. Ca2+ induces an increase in the phosphate-ATP exchange at low Mg2+ concentrations.     

Rapid release of 42K and 86Rb from an occluded state of the Na,K-pump in the presence of ATP or ADP

We have measured the time course of release of 42K and 86Rb from an occluded state of the Na,K-pump using a rapid filtration apparatus. We have found that at 20 degrees C and in the presence of ATP, 42K is released with a rate constant of approximately 45 s-1 and 86Rb with a rate constant of approximately 20 s-1; both ATP and ADP are effective at a low affinity site (Kd approximately 0.3 and 1 mM, respectively) with the rate of deocclusion being only half as great in ADP as in ATP. Mg2+ stimulates 2-fold at low concentrations probably by forming MgATP, and free Mg2+ is strongly inhibitory at high concentrations (Kd approximately 10 mM). Mg2+ also decreases the affinity for ATP, and the data are consistent with mixed type inhibition; from the analysis the dissociation constant is approximately 1 mM for the inhibitory Mg2+ and the Rb+-occluded form without ATP. The rate of 42K or 86Rb release increases monotonically with pH while ATPase activity decreases above pH 8, so that deocclusion is not rate-limiting in the overall cycle at high pH. This is reflected by a convergence of the rate of Na,K-ATPase and Na,Rb-ATPase activities at high pH and by a decrease in the observed steady-state level of the occluded 86Rb intermediate at high pH. K+, Rb+, Na+, and Cs+, but not Li+, increase the rate of 42K and 86Rb release at constant ionic strength, presumably at sites other than the transport sites. The spontaneous rate of deocclusion is only approximately 0.1 s-1 at low ionic strength in the absence of nucleotides, and it is increased markedly by all cations tested except Li+. Overall the data are consistent with deocclusion as a rate-limiting step in the Na,K-pump cycle.     

Kinetic mechanism of Fo x F1 mitochondrial ATPase: Mg2+ requirement for Mg x ATP hydrolysis

The initial rates of ATP hydrolysis catalyzed by Fo x F1 (bovine heart submitochondrial particles) preincubated in the presence of Pi for complete activation of the oligomycin-sensitive ATPase were measured as a function of ATP, Mg2+, and Mg x ATP concentrations. The results suggest the mechanism in which Mg x ATP complex is the true substrate of the ATPase and the second Mg2+ bound at a specific pH-dependent site is needed for the catalysis. Simple hyperbolic Michaelis--Menten dependences of the reaction rate on the substrate (Mg x ATP) and activating Mg2+ were found. In contrast to the generally accepted view, no inhibition of ATPase by free Mg2+ was found. Inhibition of the reaction by free ATP is due to a decrease of free Mg2+ needed for the catalysis. In the presence of both Ca2+ and Mg2+ the kinetics of ATP hydrolysis suggest that the Ca x ATP complex is neither hydrolyzed nor competes with Mg x ATP, and free Ca2+ does not affect the hydrolysis of Mg x ATP complex. A crucial role of free Mg2+ in the time-dependent inhibition of ATPase by azide is shown. The dependence of apparent Km for Mg x ATP on saturation of the Mg2+-specific site suggests the formal ping-pong mechanism in which bound Mg2+ participates in the overall reaction after dissociation of one product (most likely Pi) thus promoting either release of ADP (catalytic turnover) or slow isomerization of the enzyme--product complex (formation of the dead-end ADP(Mg2+)-inhibited enzyme). The rate of Mg x ATP hydrolysis only slightly depends on pH at saturating Mg2+. In the presence of limited amounts of free Mg2+ the pH dependence of the initial rate corresponds to the titration of a single group with pKa = 7.5. The simple competition between H+ and activating Mg2+ was observed. The specific role of Mg2+ as a coupling cation for energy transduction in Fo x F1-ATPase is discussed.