High Affinity ATP Binding Site Research Articles

N-Ethylmaleimide-sensitive Fusion Protein Contains High and Low Affinity ATP-binding Sites That Are Functionally Distinct

N-Ethylmaleimide-sensitive factor (NSF) has been shown to be involved in numerous intracellular membrane fusion events of both the regulated and constitutive secretory pathways. Sequence analysis indicates that the NSF subunit contains two nucleotide-binding sites, both with the classical Walker A and B motifs. In this report, we examine the nucleotide binding properties of NSF. The homotrimer contains three high affinity ATP-binding sites with Kd = 30-40 nM for ATP and Kd = 2 microM for ADP. This class of binding sites did not bind AMP, adenine, or GTP. A second class of lower affinity nucleotide binding sites with a Kd = 15-20 microM was also detected. Using various mutant forms of NSF, the high affinity nucleotide-binding sites were localized to the D2 domains and the low affinity sites were localized to the D1 domains. Functionally it is these lower affinity sites in D1 that are crucial for NSF activity. Nucleotide concentration greatly affected the ability of NSF to interact with alpha-SNAP.SNARE (soluble NSF attachment protein-SNAP receptor) complex, suggesting that only when the D1 domain ATP-binding sites are occupied does NSF bind to the alpha-SNAP.SNARE complex.

Stopped-flow kinetic investigations of conformational changes of pig kidney Na+,K+-ATPase.

The kinetics of Na+-dependent partial reactions of the Na+,K+-ATPase were investigated via the stopped-flow technique using the fluorescent labels RH421 and BIPM. After the enzyme is mixed with MgATP, both labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau1 approximately 180 s-1 (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3 --> E2P(Na+)3 + ADP). The rate of the phosphorylation reaction measured by the acid quenched-flow technique was 190 s-1 at 100 microM ATP, suggesting that phosphorylation controls the kinetics of the RH421 signal and that the conformational change is very fast (>/=600 s-1). The rate of the RH421 signal was optimal at pH 7.5. The Na+ concentration dependence of 1/tau1 showed half-saturation at a Na+ concentration of 8-10 mM with positive cooperativity involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high affinity ATP binding site determined from the ATP concentration dependence of 1/tau1 was 7.0 (+/-0.6) microM, while the apparent Kd for the low affinity site and the rate constant for the E2 to E1 conformational change evaluated in the absence of Mg2+ were 143 (+/-17) microM and </= 28 s-1. At RH421 concentrations in the micromolar range, a decrease in the value of 1/tau1 is observed. On the basis of rapid quenched-flow measurements, this inhibition can be attributed to a reaction step subsequent to phosphorylation. This accounts for previously observed kinetic discrepancies between RH421 and BIPM.

Identification of Arginyl Residues Located at the ATP Binding Site of Sarcoplasmic Reticulum Ca2+-ATPase: MODIFICATION WITH 1,2-CYCLOHEXANEDIONE

Sarcoplasmic reticulum vesicles were treated with 1, 2-cyclohexanedione (CHD) in sodium borate (pH 8.0). The Ca2+-ATPase activity was completely inhibited. Inhibition of Mg.ATP and Mg.ADP binding to the high affinity ATP binding site as well as inhibition of phosphorylation with ATP occurred simultaneously with the inhibition of the Ca2+-ATPase activity. Phosphorylation with acetyl phosphate was not inhibited. The Ca2+-ATPase was strongly protected by Mg.ATP, Mg.ADP, and Mg.AMP against this inhibition. Binding of acetyl phosphate or Pi to the enzyme gave no protection. Phosphorylation with acetyl phosphate also had no protective effect. Peptide mapping of the tryptic digests, detection of peptides containing CHD-modified arginyl residues with Girard's reagent T, and sequencing revealed that Arg-489, Arg-505, and Arg-678 were modified with CHD. Arg-489 and Arg-678 were almost completely protected by Mg.ATP against this modification, but partially protected by prelabeling with fluorescein 5-isothiocyanate, which occupies the adenosine binding region in the ATP binding site. In contrast, Arg-505 was slightly protected by Mg.ATP and almost completely protected by prelabeling with fluorescein 5-isothiocyanate. Taken together, these findings suggest that Arg-489 and Arg-678 are located in or near the region occupied by the triphosphate moiety of ATP, either or both of these residues being in or close to the region occupied by the alpha-phosphoryl group in the high affinity ATP binding site and involved in the CHD-induced inhibition of this enzyme and that Arg-505 is very close to (but slightly out of) the adenosine binding region in the ATP binding site. The acetyl phosphatase activity and phosphorylation with Pi were also inhibited by the CHD treatment, but the inhibitions were considerably slower than those described above. This suggests that the arginyl residues involved in these inhibitions are distinct from that involved in the inhibition of the Ca2+-ATPase activity.

Alteration in cardiac sarcolemmal ATP receptors by oxyradicals.

We previously demonstrated that cardiac sarcolemmal membranes bind [35S]ATP gamma S at both low and high affinity binding sites. In this study we examined the effects of some P2-purinoceptor antagonists as well as of two oxidants (H2O2 and HOCl) on the high affinity ATP-binding sites under in vitro conditions. It was found that putative P2-purinoceptor antagonists such as Cibacron blue, suramin, and 4,4'-diisothiocyanatostilbene 2-2 acid markedly inhibited specific ATP-binding with sarcolemmal membrane. H2O2 produced a biphasic effect (first increase and then decrease) on the specific ATP-binding with cardiac sarcolemma in a time- and concentration-dependent manner; these effects were prevented by catalase. On the other hand, HOCl markedly inhibited ATP-binding; this inhibition was prevented by l-methionine. These results suggest that the high affinity ATP-binding sites in cardiac sarcolemma may represent the P2-purinoceptors, which are susceptible to modification by oxidative stress under pathophysiological conditions including myocardial ischemia-reperfusion injury.

Characterization of a Potential Catalytic Residue, Asp-133, in the High Affinity ATP-binding Site of Escherichia coli SecA, Translocation ATPase

The high affinity ATP-binding site of SecA is located in its amino-terminal domain possessing amino acid sequences, the Walker A (GXXXXGKT) and B (ZZZZD) motifs, that are characteristic of a major class of nucleotide-binding sites (Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951). Recently, we proposed that proteins possessing a typical set of Walker A and B motifs contain a conserved Glu or Asp between the two motifs. This Glu or Asp acts as a "catalytic residue" that activates a water molecule for an in-line attack on the gamma-phosphate of ATP (Amano, T., Yoshida, M., Matsuo, Y., and Nishikawa, K.(1995) FEBS Lett. 359, 1-5). In the present study, the aspartate residue at position 133 in Escherichia coli SecA, which could be the "catalytic residue," was mutated to an asparagine. The mutant SecA (SecA D133N) protein was expressed in E. coli CK4706, encoding a duplication of the secA gene, and purified to homogeneity. The in vitro protein translocation activity and membrane vesicle stimulated ATPase activity of SecA D133N were drastically reduced. Proteolytic studies indicated that the conformational changes of the mutant SecA occurring on interaction with ATP, presecretory proteins, phospholipids, and membrane vesicles, were similar to those of wild-type SecA. The mutant SecA allowed the signal peptide cleavage of proOmpA during translocation, indicating that the mutant retains the ability to bind ATP to perform the initial step of the translocation reaction. These data indicate that the carboxyl group of Asp-133 plays a role as a catalytic carboxylate, which activates a water molecule to attack gamma-phosphate of ATP, and the mutant lacking this residue cannot perform the total translocation but can still perform the initial step of the protein translocation.

Evidence that most high-affinity ATP binding sites on aortic endothelial cells and membranes do not correspond to P 2 receptors

It has recently been demonstrated that two types of ATP receptors, the P 2 y and P 2 u receptors, are coexpressed on bovine aortic endothelial cells. The aim of the present study was to characterize directly P 2 y and P 2 u subtypes on intact bovine aortic endothelial cells and on membranes prepared from these cells using adenosine 5′-0-(3-thio[ 35S]triphosphate) ([ 35S]ATPγS). [ α- 32P]ATP and [ α- 32P]UTP as radioligands. [ 35S]ATPγS binding to bovine aortic endothelial cell membranes was saturable and apparently involved a single class of high-affinity binding sites ( K d : 14 ± 11 nM; B max 1.6 ± 0.7 pmol/mg protein; mean ± S.D.). A similar class of high-affinity binding sites was identified with [ α- 32P]ATP ( K d : 14 ± 9 nM; B max: 1.7 ± 1.1 pmol/mg protein; mean ± S.D.). Competition experiments showed that only one third of these sites bound 2-methylthio-ATP (2-MeSATP) with high affinity ( K i : 21 ± 5 and 14 ± 10 nM, mean ± S.D., for [ 35S]ATPγS and [ α- 32P]ATP, respectively) and might therefore represent the P 2 y receptors. UTP did not compete with [ 35S]ATPγS or [ α- 32P]ATP for binding at the remaining sites, indicating that they are not the P 2 u receptors. No high-affinity UTP binding sites could be detected using [ α- 32P]UTP. [ 35S]ATPγS binding to intact bovine aortic endothelial cells was competed by ATPγS ( K d : 1.0 ± 0.5 gmM; mean ± S.D.), but not by 2-MeSATP and UTP, indicating that these binding sites are neither the P 2 y nor the P 2 u receptors.

Integration of SecA protein into the Escherichia coli inner membrane is regulated by its amino-terminal ATP-binding domain.

SecA protein, the ATPase promoting translocation of proteins across the Escherichia coil inner membrane, contains two ATP-binding domains that differ greatly in their affinity for bound nucleotide. In order to define more precisely the location of the high-affinity nucleotide-binding site, oligonucleotide-directed mutagenesis was used to introduce cysteine residues into the SecA sequence, and a cysteine-specific cleavage reagent was employed to generate defined peptides of SecA protein after photocross-linking with [alpha-(32)P]-ATP. This analysis revealed that the nucleotide was cross-linked between amino acid residues 75 and 97 of SecA protein. The biochemical function of the high affinity ATP-binding domain was explored by subcellular fractionation studies which demonstrated that SecA proteins defective in this region were found almost exclusively in their integral membrane form, while SecA proteins with defects in the low-affinity ATP-domain showed a normal distribution of cytosolic, peripheral and integral membrane forms. Interestingly, the SecA51(Ts) protein that has a Leu to Pro substitution at amino acid residue 43 bound ATP with high affinity, but its fractionation pattern and translocation ATPase activity were similar to those of proteins with defects in the high-affinity ATP-binding site. These results delimit more precisely the high-affinity ATP-binding domain of SecA, indicate the importance of the early amino-terminal region of SecA protein in the functioning of this domain, and demonstrate the role of this domain in regulating penetration of SecA protein into the inner membrane. Our results lead to a simple model for the regulation of a cycle of SecA insertion into, and de-insertion from, the inner membrane by the activity of the high affinity ATP-binding domain.

Oligomeric Regulation of Gastric H+,K+-ATPase

The H+,K+-ATPase of intact gastric vesicles has two Km values for ATP hydrolysis, 7 and 80 microM. Irradiation of vesicles with ultraviolet light in the presence of 1 mM ATP resulted in K+-ATPase activity that shows only the low affinity ATP binding. The irradiation stimulated or inhibited proton uptake rate compared with control vesicles at high or low ATP concentrations, respectively. The relation between proton uptake rate and K+-ATPase activity at different ATP concentrations was linear with irradiated vesicles and nonlinear with control vesicles. These results indicate that hydrolysis at the high affinity ATP binding site regulates the energy-transport coupling in negative and positive manners at high and low ATP concentrations, respectively. The complete inhibition of K+-ATPase by a specific proton pump inhibitor E3810 (rabeprazole) (2-([4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methylsulf i nyl)-1H-benzimidazole sodium salt) occurred when E3810 bound to half of the alpha-subunit of H+,K+-ATPase in unirradiated vesicles at both 200 and 10 microM ATP, whereas the complete inhibition of proton uptake occurred when E3810 bound to half or a quarter of the alpha-subunit at 200 or 10 microM ATP, respectively. These results suggest that dimeric interaction between the alpha-subunits is necessary for the enzyme activity at all ATP concentrations and that dimeric or tetrameric interaction is necessary for proton transport at high or low ATP concentrations, respectively.

The Ubiquitous VA68 Isoform of Subunit-A of the Vacuolar H +-ATPase Is Highly Expressed in Human Osteoclasts

The 67-kDa subunit A of the vacuolar-type H +-ATPase carries the high affinity ATP binding site and together with the 57-kDa subunit B forms the catalytic domain. Two isoforms of subunit A, VA68 and HO68, were cloned from an osteoclastoma cDNA library. We have analyzed their respective expression patterns in different tissues by RNAse A protection and in situ hybridization. The HO68 isoform was found to be present only in the tumor originally used to construct the cDNA library, whereas the ubiquitous VA68 RNA isoform was detected in large osteoclastic cells, as well as in brain and kidney, by RNAse protection assay. Furthermore, we localised the strong signal observed in osteoclastoma RNA to the large osteoclastic cells by in situ hybridisation. These findings suggest that the subunit A highly expressed in human osteoclasts is the ubiquitous isoform, VA68.

Identification of the Magnesium-binding Domain of the High-affinity ATP-binding Site of the Bacillus subtilis and Escherichia coli SecA Protein

The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane. The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA. This domain contains a tandem repeat of the "so-called" Walker B-motif, hXhhD (Walker, J.E., Saraste, M., Runswick, M.J., and Gay, N.J. (1982) EMBO J. 1, 945-951), that in combination with motif A is responsible for the Mg(2+)-phosphate protein interaction. Two aspartate residues at positions 207 and 215 of the Bacillus subtilis SecA, and Asp-217 in the Escherichia coli SecA, that could be Mg2+ ion ligands, were individually mutated to an asparagine. Mutant SecA proteins were unable to growth-complement an E. coli secA amber mutant strain, and the E. coli SecA mutant interfered with the translocation of precursor proteins in vivo. B. subtilis mutant SecA proteins were expressed to a high level and purified to homogeneity. The high affinity ATP and Mg(2+)-ion binding activity was reduced in the Asp-207 mutant, and completely lost in the Asp-215 mutant. Both SecA proteins were defective in lipid-stimulated ATPase activity. Proteolytic studies suggest that the two subunits of the mutated dimeric SecA proteins are present in different conformational states. These data suggest that Asp-207 and Asp-215 are involved in the binding of the Mg(2+)-ion when Mg(2+)-ATP is bound to SecA, while Asp-207 fulfills an additional catalytic role, possibly in accepting a proton during catalysis.

An extra phosphorylation of Na+,K(+)-ATPase by paranitrophenylphosphate (pNPP): evidence for the oligomeric nature of the enzyme.

Paranitrophenylphosphate (pNPP) induced fluorescence changes in fluorescence isothiocyanate (FITC)-labeled Na+,K(+)-ATPase preparations. The extents of changes were similar to those induced by acetylphosphate (AcP) accompanying accumulation of a K(+)-sensitive phosphoenzyme (E2P) and an ouabain bound phosphoenzyme in the presence of Mg2+ and 16 mM Na+. Phosphoenzymes formed from [32P]pNPP were shown to turn over. The ratio of the maximum amount of the phosphoenzyme formed from pNPP to that of the phosphoenzyme formed from ATP and that of the ouabain-enzyme complex under steady-state conditions was shown to be close to 1:0.5:1. Such extra phosphorylation has hitherto only been observed in a transient state with the additions of high concentrations of ATP [Peluffo, R.D., Garrahan, P.J., and Rega, A. (1992) J. Biol. Chem. 267, 6596-6601]. Our data are compatible with the simultaneous presence of high and low affinity ATP-binding sites in Na+,K(+)-ATPase [Hamer, E. and Schoner, W. (1993) Eur. J. Biochem. 213, 743-748]. The maximum amount of paranitrophenol-sensitive fraction to synthesize [32P]pNPP in fully accumulated ADP-sensitive phosphoenzyme (E1P) from [32P]ATP was around 1/4 of the amount of ouabain-enzyme complex. These data and others indicate that a much higher degree of oligomerization, rather than (alpha beta)2, may be the functional unit of the enzyme in the membranes.

Stoichiometry of tight binding of magnesium and fluoride to phosphorylation and high-affinity binding of ATP, vanadate, and calcium in the sarcoplasmic reticulum Ca(2+)-ATPase.

We previously showed that, when the purified Ca(2+)-ATPase from sarcoplasmic reticulum (SR) is treated with fluoride (F-) in the presence of Mg2+, a complete inactivation of the enzyme is induced by tight binding of approximately 2 mol of Mg2+ and 4 mol of F- to the catalytic site per mole of phosphorylation site [Kubota, T., Daiho, T., & Kanazawa, T. (1993) Biochim. Biophys. Acta 1163, 131-143]. Contradictorily, on the basis of the postulated content of the Ca(2+)-ATPase in F(-)-treated SR vesicles, Coll and Murphy [(1992) J. Biol. Chem. 267, 21584-21587] suggested that each inactivated enzyme contains one tightly-bound Mg2+ and two tightly-bound F-. The present study has been made to resolve this conflict. The contents of phosphorylation site, high-affinity ATP-binding site, high-affinity vanadate-binding site, and high-affinity Ca(2+)-binding site in the SR vesicles used were 3.33 +/- 0.06, 3.54 +/- 0.12, 3.34 +/- 0.04, and 6.98 +/- 0.16 nmol/mg, respectively. When the vesicles were incubated with F- in the presence of Mg2+, the Ca(2+)-ATPase was inactivated progressively. After removal of unbound Mg2+ and F- by gel filtration, tightly-bound Mg2+ and F- were determined by use of an atomic absorption spectrophotometer and a F(-)-selective electrode. A linear relationship existed between the extent of the enzyme inactivation and the contents of the tightly-bound ligands. The contents of tightly-bound Mg2+ and F- in the fully inactivated vesicles were 6.65 and 12.6 nmol/mg, respectively. The same stoichiometry was obtained with another preparation of SR vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Lysine 155 in beta-subunit is a catalytic residue of Escherichia coli F1 ATPase.

The "homology A" ("glycine-rich" or "P-loop") consensus sequence occurs in the catalytic sites of F1F0 ATP synthase enzymes. The conserved lysine of this motif is beta-subunit Lys-155 in Escherichia coli F1. The role of this lysine in binding and catalysis at the high affinity ATP binding site was studied with the mutants beta K155Q and beta K155E by measuring the rates of ATP binding/release, ATP hydrolysis/synthesis, and Pi release as a function of pH varied from 5.5 to 9.5. In wild type, protonated beta Lys-155 appears to contribute significantly to high affinity binding of ATP, probably through hydrogen bonding to the gamma-phosphate. ATP hydrolysis and synthesis were impaired strongly in the mutants, and the reaction equilibrium constant, which was pH-independent in wild type, was highly pH-dependent in beta K155Q and beta K155E. Studies of steady-state ATPase turnover showed that positive catalytic cooperativity was virtually absent and the pH-dependent component of positive catalytic cooperativity was abolished or reversed in the mutants. The data demonstrate that residue beta K155 is a critical catalytic residue in F1 ATPase.

Nucleotide specificity of cardiac sarcoplasmic reticulum. Inhibition of GTPase activity by ATP analogue in fluorescein isothiocyanate-modified calcium ATPase

Unlike skeletal muscle sarcoplasmic reticulum, canine cardiac sarcoplasmic reticulum hydrolyzes GTP in ways that are similar and different from ATP hydrolysis. Also, ATP and ATP analogues inhibit GTPase activity noncompetitively with a Ki compatible with the high affinity ATP-binding site (c.f. Tate, C.A., Bick, R.J., Blaylock, S., Youker, K., Scherer, N.M., and Entman, M.L. (1989) J. Biol. Chem. 264, 7809-7813). This suggested that ATP and GTP may enter the reaction pathway at separate nucleotide-binding sites on the CaATPase. To test this hypothesis, cardiac sarcoplasmic reticulum was incorporated with fluorescein isothiocyanate (FITC), which apparently binds at or near the ATP-binding site of the enzyme, preventing ATP binding. After FITC incorporation, calcium-dependent ATPase activity, but not GTPase activity, was completely inhibited. Adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), but not guanyl-5'-yl imidodiphosphate, protected against FITC incorporation and the inhibition of calcium-dependent ATPase activity; at least 100 microM AMP-P(NH)P was required for some protection. Despite FITC incorporation, AMP-P(NH)P still inhibited the GTPase activity with a Ki of 3-7 microM. Direct photo-affinity labeling with either 0.2 microM [alpha-32P]ATP or 0.2 microM [alpha-32P]GTP demonstrated that FITC incorporation did not prevent ATP or GTP binding. The mechanism of FITC inhibition of calcium-dependent ATPase activity was related to the prevention of all calcium-dependent, but not calcium-independent, reactions with both nucleotides.

Phosphate binding and ATP‐binding sites coexist in Na+/K+‐transporting ATPase, as demonstrated by the inactivating MgPO4 complex analogue Co(NH3)4PO4

Tetrammine cobalt(III) phosphate [Co(NH3)4PO4] inactivates Na+/K(+)-ATPase in the E2 conformational state, dependent on time and concentration, according to Eqn (1): Co(NH3)4PO4 + E2 Kd in equilibrium E2.Co(NH3)4PO4k2----E'2.Co(NH3)4PO4. The inactivation rate constant k2 for the formation of a stable E'2.Co(NH3)4PO4 at 37 degrees C was 0.057 min-1; the dissociation constant, Kd = 300 microM. The activation energy for the inactivation process was 149 kJ/mol. ATP and the uncleavable adenosine 5'-[beta, gamma-methylene]triphosphate competed with Co(NH3)4PO4 for its binding site with Ks = 0.41 mM and 5 mM, respectively. MgPO4 competed with Co(NH3)4PO4 linearly, with Ks = 50 microM, as did phosphate (Ks = 16 mM) and Mg2+ (Ks = 160 microM). It is concluded that the MgPO4 analogue binds to the MgPO4-binding subsite of the low-affinity ATP-binding site (of the E2 conformation). Also, Na+ (Ks = 860 microM) protected the enzyme against inactivation in a competitive manner. From the intersecting (slope and intercept linear) noncompetitive effect of Na+ against the inactivation by Co(NH3)4PO4, apparent affinities of K+ for the free enzyme of 41 microM, and for the E.Co(NH3)4PO4 complex of 720 microM, were calculated. Binding of Co(NH3)4PO4 to the enzyme inactivated Na+/K(+)-ATPase and K(+)-activated phosphatase, and, moreover, prevented the occlusion of 86Rb+; however, the activity of the Na(+)-ATPase, the phosphorylation capacity of the high-affinity ATP-binding site and the ATP/ADP-exchange reaction remained unchanged. With Co(NH3)432PO4 a binding capacity of 135 pmol unit enzyme was found. Phosphorylation and complete inactivation of the enzyme with Co(NH3)432PO4 or the 32P-labelled tetramminecobalt ATP ([gamma-32P]Co(NH3)4ATP) at the low-affinity ATP-binding site, allowed (independent of the purity of the Na+/K(+)-ATPase preparation) a further incorporation of radioactivity from 32P-labelled tetraaquachromium(III) ATP ([gamma-32P]CrATP) to the high-affinity ATP-binding site with unchanged phosphorylation capacity. However, inactivation and phosphorylation of Na+/K(+)-ATPase by [gamma-32P]CrATP prevented the binding of Co(NH3)4 32PO4 or [gamma-32P]Co(NH3)4ATP to the enzyme. [gamma-32P]CO(NH3)4ATP and Co(NH3)432PO4 are mutually exclusive. The data are consistent with the assumption of a cooperation of catalytic subunits within an (alpha,beta)2-diprotomer, which change their interactions during the Na+/K(+)-pumping process. Our findings seem not to support a symmetrical Repke and Stein model of enzyme action.

Blocking of Na+/K+ transport by the MgPO4 complex analogue Co(NH3)4PO4 leaves the Na+/Na+‐exchange reaction of the sodium pump unaltered and shifts its high‐affinity ATP‐binding site to a Na+‐like form

Inactivation of Na+/K(+)-ATPase activity by the MgPO4 complex analogue Co(NH3)4PO4 leads, in everted red blood cell vesicles, to the parallel inactivation of 22Na+/K+ flux and 86Rb/Rb+ exchange, but leaves the 22Na+/Na(+)-exchange activity and the uncoupled ATP-supported 22Na+ transport unaffected. Furthermore, inactivation of purified Na+/K(+)-ATPase by Co(NH3)4PO4 leads to a parallel decrease of the capacity of the [3H]ouabain receptor site, when binding was studied by the Mg2+/Pi-supported pathway (ouabain-enzyme complex II) but the capacity of the ouabain receptor site was unaltered, when the Na+/Mg2+/ATP-supported pathway (ouabain-enzyme complex I) was used. No change in the dissociation constants of either ouabain receptor complex was observed following inactivation of Na+/K(+)-ATPase. When eosin was used as a marker for the high-affinity ATP-binding site of the E1 conformation, formation of stable E'2.Co(NH3)4PO4 complex led to a shift in the high-affinity ATP-binding site towards the sodium form. This led to an increase in the dissociation constant of the enzyme complex with K+, from 1.4 mM with the unmodified enzyme to 280 mM with the Co(NH3)4PO4-inactivated enzyme. It was concluded, that the effects of Co(NH3)4PO4 on the partial activities of the sodium pump are difficult to reconcile with an alpha, beta-protomeric enzyme working according the Albers-Post scheme. The data are consistent with an alpha 2, beta 2 diprotomeric enzyme of interacting catalytic subunits working with a modified version of the Albers-Post model.

How do MgATP analogues differentially modify high‐affinity and low‐affinity ATP binding sites of Na+/K+ ‐ATPase?

The exchange-inert tetra-ammino-chromium complex of ATP [Cr(NH3)4ATP], unlike the analogous cobalt complex Co(NH3)4ATP, inactivated Na+/K(+)-ATPase slowly by interacting with the high-affinity ATP binding site. The inactivation proceeded at 37 degrees C with an inactivation rate constant of 1.34 x 10(-3) min-1 and with a dissociation constant of 0.62 microM. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na+/K(+)-ATPase by Cr(NH3)4ATP, and its H2O-substituted derivatives Cr(NH3)3(H2O)ATP, Cr(NH3)2(H2O)2ATP and Cr(H2O)4ATP was carried out. The substitution of the H2O ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H2O)4ATP was 29-fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(III) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme's active-site residues does not seem to play a significant role in the inactivation of Na+/K(+)-ATPase by Cr(III)-ATP complexes. Inactivation of the enzyme by Rh(H2O)nATP occurred by binding of this analogue to the high-affinity ATP site with an apparent dissociation constant of 1.8 microM. The observed inactivation rate constant of 2.11 x 10(-3) min-1 became higher when Na+ or Mg2+ or both were present. The presence of K+ however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na+ reactivated the Rh(H2O)nATP-inactivated enzyme. Co(NH3)4ATP inactivates Na+/K(+)-ATPase by binding to the low-affinity ATP binding site only at high concentrations. However, inactivation of the enzyme by Cr(III)-ATP or Rh(III)-ATP complexes was prevented when low concentrations of Co(NH3)4ATP were present. This indicates that, although Co(NH3)4ATP interacts with both ATP sites, inactivation occurs only through the low-affinity ATP site. Inactivation of Na+/K(+)-ATPase was faster by the delta isomer of Co(NH3)4ATP than by the delta isomer. Co(NH3)4ATP, but not Cr(H2O)4ATP or adenosine 5'-[beta,gamma-methylene]triphosphate competitively inhibited K(+)-activated p-nitrophenylphosphatase activity of Na+/K(+)-ATPase, which is assumed to be a partial reaction of the enzyme catalyzed by the low-affinity ATP binding site.

Shift to the Na+ from of Na+/K+‐transporting ATPase due to modification of the low‐affinity ATP‐binding site by Co(NH3)4ATP

1. Inactivation of purified Na+/K+-transporting ATPase by the MgATP complex analogue Co(NH3)4ATP, which binds to the low-affinity ATP-binding site, results in the concomitant inhibition of the K+-activated p-nitrophenylphosphatase, which is considered to be a partial reaction catalyzed by the enzyme in the E2 conformational state. 2. Complete inactivation of Na+/K+-transporting ATPase by Co(NH3)4ATP does not alter the ADP/ATP exchange reaction which is considered to be part of the catalytic activity in the E1 conformation. 3. The enzyme binds eosin at the high-affinity ATP-binding site as measured by the change in eosin fluorescence. Eosin binding to the Co(NH3)4ATP-inactivated enzyme is, in contrast to the untreated enzyme, not stimulated by Na1. Inactivation by Co(NH3)4ATP increased the half-maximal opposing effect of K+ on eosin binding from 1.1 mM in the control to 43.2 mM in the almost completely inactive enzyme. No eosin fluorescence changes were observed when the Co(NH3)4ATP-inactivated enzyme was treated subsequently with CrATP. This MgATP complex analogue forms a stable complex at the high-affinity ATP-binding site. CrATP thus abolishes eosin binding. 4. It is concluded, that Co(NH3)4ATP interacts with Na+/K+-transporting ATPase in the E2 conformation and arrests it there. This affects eosin binding to the high-affinity ATP-binding site, since the K+ sensitivity is lost. A possible interpretation of these differing effects of Co(NH3)4ATP on partial reactions of Na+/K+-transporting ATPase is that the sodium pump works as an (alpha,beta)2 diprotomer. It is likely that the arrest of one alpha,beta promoter in the E2 conformational state by occupancy of the low-affinity ATP-binding site with Co(NH3)4ATP induces the Na+ form (E1 form) in the corresponding alpha,beta promoter, as is indicated by the unaffected ADP/ATP exchange and the response of the eosin fluorescence on Na+ and K+.

Inhibition and labeling of sodium, potassium ATPase by the dialdehyde derivative of ATP

Canine renal Na, K-ATPase was treated with ATP dialdehyde, “oxATP” (20 μ m), as described by G. Ponzio B. Rossi, and M. Lazdunski (1983, J. Biol. Chem. 258, 8201–8205). In this system, a by-product, formaldehyde, was the inactivator. We modified the system to minimize such inhibition and to speed up the reaction. oxATP itself inactivated the enzyme at a rate that was slow at first and later speeded up. We fitted a precursorproduct model to the data. Labeling with [ 3H]oxATP indicated about three sites per α β protomer at complete inactivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled enzyme showed radioactivity in many components, in the α and β subunits and in small molecules at the tracker dye region. ATP (20 m m) prevented all labeling and inactivation. Ponzio et al. concluded that oxATP labels covalently an ATP binding site. Our experiments did not support this conclusion. Ouabain did not affect labeling. Sodium stimulated both inhibition and labeling more than potassium did, indicating a high-affinity ATP binding site, if any. But nucleotide specificity for preventing or producing inhibition did not correspond to nucleotide specificity for binding of ATP to the native enzyme. Blocking the ATP binding center with fluorescein isothiocyanate or fluorosulfonyl benzoyl adenosine had no effect on [ 3H]oxATP labeling. ATP also prevented [ 3H]oxATP labeling of bovine serum albumin or of integral-membrane proteins.

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.