Nucleotide Binding to Na,K-ATPase: p K Values of the Groups Affecting the High Affinity Site † (original) (raw)
Related papers
Nucleotide Binding to Na,K-ATPase: The Role of Electrostatic Interactions †
Biochemistry, 2002
The contribution of electrostatic forces to the interaction of Na,K-ATPase with adenine nucleotides was investigated by studying the effect of ionic strength on nucleotide binding. At pH 7.0 and 20°C, there was a qualitative correlation between the equilibrium dissociation constant (K d) values for ATP, ADP, and MgADP and their total charges. All K d values increased with increasing ionic strength. According to the Debye-Hückel theory, this suggests that the nucleotide binding site and its ligands have "effective" charges of opposite signs. However, quantitative analysis of the dependence on ionic strength shows that the product of the effective electrostatic charges on the ligand and the binding site is the same for all nucleotides, and is therefore independent of the total charge of the nucleotide. The data suggest that association of nucleotides with Na,K-ATPase is governed by a partial charge rather than the total charge of the nucleotide. This charge, interacting with positive charges on the protein, is probably the one corresponding to the R-phosphate of the nucleotide. Dissociation rate constants measured in complementary transient kinetic experiments were 13 s-1 for ATP and 27 s-1 for ADP, independent of the ionic strength in the range 0.1-0.5 M. This implies similar association rate constants for the two nucleotides (about 40 × 10 6 M-1 s-1 at I) 0.1 M). The results suggest that long-range Coulombic forces, affecting association rates, are not the main contributors to the observed differences in affinities, and that local interactions, affecting dissociation rates, may play an even greater role.
Neutralization of the Charge on Asp369 of Na+,K+-ATPase Triggers E1 E2 Conformational Changes
Journal of Biological Chemistry, 2009
This work investigates the role of charge of the phosphorylated aspartate, Asp 369 , of Na ؉ ,K ؉-ATPase on E 1 7 E 2 conformational changes. Wild type (porcine ␣ 1 /His 10- 1), D369N/ D369A/D369E, and T212A mutants were expressed in Pichia pastoris, labeled with fluorescein 5-isothiocyanate (FITC), and purified. Conformational changes of wild type and mutant proteins were analyzed using fluorescein fluorescence (Karlish, S. J. (1980) J. Bioenerg. Biomembr. 12, 111-136). One central finding is that the D369N/D369A mutants are strongly stabilized in E 2 compared with wild type and D369E or T212A mutants. Stabilization of E 2 (Rb) is detected by a reduced K 0.5 Rb for the Rb ؉induced E 1 7 E 2 (2Rb) transition. The mechanism involves a greatly reduced rate of E 2 (2Rb) 3 E 1 Na with no effect on E 1 3 E 2 (2Rb). Lowering the pH from 7.5 to 5.5 strongly stabilizes wild type in E 2 but affects the D369N mutant only weakly. Thus, this "Bohr" effect of pH on E 1 7 E 2 is due largely to protonation of Asp 369. Two novel effects of phosphate and vanadate were observed with the D369N/D369A mutants as follows. (a) E 1 3 E 2 ⅐P is induced by phosphate without Mg 2؉ ions by contrast with wild type, which requires Mg 2؉. (b) Both phosphate and vanadate induce rapid E 1 3 E 2 transitions compared with slow rates for the wild type. With reference to crystal structures of Ca 2؉-ATPase and Na ؉ ,K ؉-ATPase, negatively charged Asp 369 favors disengagement of the A domain from N and P domains (E 1), whereas the neutral D369N/D369A mutants favor association of the A domain (TGES sequence) with P and N domains (E 2). Changes in charge interactions of Asp 369 may play an important role in triggering E 1 P(3Na) 7 E 2 P and E 2 (2K) 3 E 1 Na transitions in native Na ؉ ,K ؉-ATPase.
Fluorescence measurements of nucleotide association with the Na+/K+-ATPase
Biochimica et Biophysica …, 2009
The Na + /K + -ATPase, a membrane-associated ion pump, uses energy from the hydrolysis of ATP to pump 3 Na + ions out of and 2 K + into cells. The dependence of ATP hydrolysis on ATP concentration was measured using a fluorescence coupled-enzyme assay. The dependence on concentration of nucleotide association with the ATPase was examined using ADP and ATP-induced quenching of the fluorescence of ATPase labeled with Cy3-maleimide (Cy3-ATPase) or Alexa Fluor 546 carboxylic acid, succinimidyl ester (AF-ATPase). The kinetics of ATP hydrolysis in the presence of Na + and K + exhibited negative cooperativity with a Hill coefficient (n H ) of 0.66 and a half-maximal concentration (K 0.5 ) of 61 μM; in the absence of K + , n H was 0.58 and K 0.5 was 13 μM. Nucleotide-induced fluorescence quenching exhibited negative cooperativity with an n H of 0.3-0.5. These results suggest that negative cooperativity observed in ATP hydrolysis is attributable to negative cooperativity in nucleotide association to the ATPase. Interaction between AF-ATPase and ATP labeled with Alexa Fluor 647 (AF-ATP) showed significant Förster resonance energy transfer (FRET). These results indicate that the ATPase exists as oligoprotomeric complexes in this preparation, and that this aggregation has significant effects on enzyme function.
Specificity of nucleotide binding and coupled reactions utilising the mitochondrial ATPase
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1978
1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined, iso-Guanosine, 2'-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site. 4. The nucleotide specificities of 'coupled processes' nucleoside triphos-phate<triven reversal of electron transfer, nucleoside triphosphate-32P i exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1991
The effects of K +, Na + and nucleotides (ATP or ADP) on the steady-state phosphorylafion from [3ZP]P i (0.5 and | mM) and acetyi [a~P]phosphate (AeP) (5 raM) were studied in membrane fragments and in proteofiposomes with partially purified pig kidney Na,K-ATPase incorporated. The experiments were carried out at 20°C and pH 7.0. In broken membranes, the Pi-induced phosphoenzyme levels were reduced to 40% by 10 mM K + and to 20% by 10 mM K + plus I mM ADP (or ATP); in the presence of 50 mM Na+, no E-P formation was detected. On the other hand, with AcP, the E-P formation was reduced by 10 mM K + but was 30% incre&sed by 50 mM Na+. In proteofiposomes E-P Naext, (ii) about 50% reduced by 5, 10 or 100 formation from Pi was (i) not influenced by 5-10 mM K~ or 100 mM + m]VI Ke+t and (iii) completely prevented by 50 mM + Na,~. Enzyme phosphorylation from AcP was 30% increased by 10 K~ or 50 mM Na,wt; these E-P were 50% redneed by 10-100 mM Kex t. However, E-P formed from AcP without mM 4. 4. 4-Key t or Nac~ t was not affected by extraceHular K4-. nuor~ence changes of fluorescein isothiocyanate labelled membrane fragments, indicated that E-P from AcP corresponded to an E z state in the presence of 10 mM Na 4-or 2 mM K 4-but to an El state in the absence of both cations. With pNPP, the data indicated an E~ state in the absence of Na + and K 4. and also in the presence of 20 mM Na+, and an E 2 form in the presence of 5 raM K 4-. These results suggest that, although with some slmHarities, the reversible P~ phosphorylation and the phosphatase activity of the Na,K-ATPase do not share the whole reaction pathway.
ATP-binding is stabilized by a stacking interaction within the binding site of Na+/K+-ATPase
Biochemical and Biophysical Research Communications, 2003
Site-directed mutagenesis was applied to modify phenylalanines (Phe 475 Trp, Phe 548 Tyr, and both) to generate mutants on the basis of molecular modeling of the ATP-binding domain of Na þ /K þ-ATPase, in order to characterize the forces that stabilize ATP in its binding pocket. Each of the mutants was examined by Raman difference spectroscopy, i.e., as a difference between the spectrum of the domain with and without bound ATP. It was shown that Phe 475 plays a key role in stabilizing ATP-binding by a stacking interaction. Phe 548 co-stabilizes ATP on the opposite site of the binding pocket and its type of interaction with ATP-binding differs from that of Phe 475 .
Microenvironment of the high affinity ATP-binding site of Na+/K+-ATPase is slightly acidic
Fluorescein-5-isothiocyanate (FITC) was used to study the high-affinity ATP-binding site of Na ؉ /K ؉ -ATPase. The molar ratio of specifically bound FITC per ␣-subunit of Na ؉ /K ؉ -ATPase was found to be 0.5 as followed from pretreatment experiments with another specific E 1 ATP-inhibitor Cr(H 2 O) 4 AdoPP[CH 2 ]P. This indicated an existence of one high affinity ATPbinding site (E 1 ATP-binding site) in the native (␣) 2diprotomer of Na ؉ /K ؉ -ATPase. Fluorescence dualexcitation ratio of specifically bound FITC revealed that at external pH 7.5, the pH value inside the E 1 ATPbinding site is 6.95 ؎ 0.18. In addition, FITC fluorescence quenching by anti-fluorescein and by iodide choline indicated the limited access of water into the small pocket of the E 1 ATP-binding site.
European Journal of Biochemistry, 1991
The acid-base properties of the nucleic base residues of ITP, GTP, and ATP, and for comparison also as far as possible of the corresponding nucleosides, were studied in dependence on their concentration, i.e. on the effect of self-association. From the dependence between the 1H-NMR chemical shifts of H-2 (where applicable), H-8, and H-1', and the pD of the solutions, the acidity constants for the deprotonation of the D+(N-7) site in D2(ITP)2-, D2(GTP)2-, D(Ino)+, and D(Guo)+, and of the D+(N-1) site in D2(ATP)2- and D(Ado)+ were calculated. Chemical shift/pD profiles for a whole series of varying concentrations of the nucleic base derivatives (= N) were constructed, including those for infinite dilution (delta o), which give the acidity constant for the monomeric N, and for infinitely concentrated solutions (delta infinity), which give the acidity constant of an N in an infinitely long stack. The acidity constants determined from the delta o/pD plots are in excellent agreement with the pKa values measured by potentiometric pH titrations of highly diluted solutions of N. The effects of self-association are striking; e.g. the pKa value of the D+(N-7) site in D2(GTP)2- is lowered by about 1 (as calculated from the delta o/pD and delta infinity/pD profiles), while the pKa value of the D+(N-1) site in D2(ATP)2- is increased by approximately 0.3; i.e. in the first case deprotonation is facilitated and in the second it is inhibited. The increasing inhibition of the H+(N-1) deprotonation with an increasing ATP concentration is due to the high stability of the dimeric [H2(ATP)]2(4-) stack for which the intermolecular H+(N-1)/gamma-P(OH)(O)2- ion pairs between the two ATP molecules are crucial. In those cases where no other significant interaction but aromatic-ring stacking in the self-association process occurs, the release of protons from protonated nitrogen-ring sites is facilitated with increasing stacking; this holds not only for D2(GTP)2- as indicated above, but also for D2(ITP)2-, D(Ino)+, and D(Ado)+. The latter example especially suggests that the situation for the D2(ATP)2- system is exceptional. Some consequences of the considered acid-base properties for biological systems are indicated.
ATP hydrolysis by Na ؉ /K ؉ -ATPase proceeds via the interaction of simultaneously existing and cooperating high (E 1 ATP) and low (E 2 ATP) substrate binding sites. It is unclear whether both ATP sites reside on the same or on different catalytic ␣-subunits. To answer this question, we looked for a fluorescent label for the E 2 ATP site that would be suitable for distance measurements by Fö rster energy transfer after affinity labeling of the E 1 ATP site by fluorescein 5-isothiocyanate (FITC). Erythrosin 5-isothiocyanate (ErITC) inactivated, in an E 1 ATP site-blocked enzyme (by FITC), the residual activity of the E 2 ATP site, namely K ؉ -activated p-nitrophenylphosphatase in a concentration-dependent way that was ATP-protectable. The molar ratios of FITC/␣-subunit of 0.6 and of ErITC/␣-subunit of 0.48 indicate 2 ATP sites per (␣) 2 diprotomer. Measurements of Fö rster energy transfer between the FITC-labeled E 1 ATP and the ErITC-labeled or Co(NH 3 ) 4 ATP-inactivated E 2 ATP sites gave a distance of 6.45 ؎ 0.64 nm. This distance excludes 2 ATP sites per ␣-subunit since the diameter of ␣ is 4 -5 nm. Fö rster energy transfer between cardiac glycoside binding sites labeled with anthroylouabain and fluoresceinylethylenediamino ouabain gave a distance of 4.9 ؎ 0.5 nm. Hence all data are consistent with the hypothesis that Na ؉ /K ؉ -ATPase in cellular membranes is an (␣) 2 diprotomer and works as a functional dimer (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321).