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Tight Binding of Bulky Fluorescent Derivatives of Adenosine to the Low Affinity E2ATP Site Leads to Inhibition of Na+/K+-ATPase

1999; Elsevier BV; Volume: 274; Issue: 4 Linguagem: Inglês

10.1074/jbc.274.4.1971

ISSN

1083-351X

Autores

Detlef Thoenges, Evžen Amler, Thomas Eckert, Wilhelm Schoner,

Tópico(s)

Ion Transport and Channel Regulation

Resumo

A Koshland-Némethy-Filmer model of two cooperating ATP sites has previously been shown to explain the kinetics of inhibition of Na+/K+-ATPase (EC3.6.1.37) by dansylated ATP (Thoenges, D., and Schoner, W. (1997)J. Biol. Chem. 272, 16315–16321). The present work demonstrates that this model adequately describes all types of interactions and kinetics of a number of ATP analogs that differ in their cooperativity of the high and low affinity ATP binding sites of the enzyme. 2′,3′-O(2,4,6-trinitrophenyl)ATP binds in a negative cooperative way to the E1ATP site (K d = 0.7 μm) and to the E2ATP site (K d = 210 μm), but 3′(2′)-O-methylanthraniloyl-ATP in a positive cooperative way with a lower affinity to the E1ATP binding site (K d = 200 μm) than to the E2ATP binding site (K d = 80 μm). 3′(2′)-O(5-Fluor-2,4-dinitrophenyl)-ATP, however, binds in a noncooperative way, with equal affinities to both ATP binding sites (K d = 10 μm). In a research for the structural parameters determining ATP site specificity and cooperativity, we became aware that structural flexibility of ribose is necessary for catalysis. Moreover, puckering of the ring atoms in the ribose is essential for the interaction between ATP sites in Na+/K+-ATPase. A number of derivatives of 2′(3′)-O-adenosine with bulky fluorescent substitutes bind with high affinity to the E2ATP site and inhibit Na+/K+-ATPase activity. Evidently, an increased number of interactions of such a bulky adenosine with the enzyme protein tightens binding to the E2ATP site. A Koshland-Némethy-Filmer model of two cooperating ATP sites has previously been shown to explain the kinetics of inhibition of Na+/K+-ATPase (EC3.6.1.37) by dansylated ATP (Thoenges, D., and Schoner, W. (1997)J. Biol. Chem. 272, 16315–16321). The present work demonstrates that this model adequately describes all types of interactions and kinetics of a number of ATP analogs that differ in their cooperativity of the high and low affinity ATP binding sites of the enzyme. 2′,3′-O(2,4,6-trinitrophenyl)ATP binds in a negative cooperative way to the E1ATP site (K d = 0.7 μm) and to the E2ATP site (K d = 210 μm), but 3′(2′)-O-methylanthraniloyl-ATP in a positive cooperative way with a lower affinity to the E1ATP binding site (K d = 200 μm) than to the E2ATP binding site (K d = 80 μm). 3′(2′)-O(5-Fluor-2,4-dinitrophenyl)-ATP, however, binds in a noncooperative way, with equal affinities to both ATP binding sites (K d = 10 μm). In a research for the structural parameters determining ATP site specificity and cooperativity, we became aware that structural flexibility of ribose is necessary for catalysis. Moreover, puckering of the ring atoms in the ribose is essential for the interaction between ATP sites in Na+/K+-ATPase. A number of derivatives of 2′(3′)-O-adenosine with bulky fluorescent substitutes bind with high affinity to the E2ATP site and inhibit Na+/K+-ATPase activity. Evidently, an increased number of interactions of such a bulky adenosine with the enzyme protein tightens binding to the E2ATP site. Cr(H20)4ATP-sensitive site with high affinity for ATP Co(NH3)4ATP-sensitive site with low affinity for ATP 2′,3′-O(2,4,6-trinitrophenyl)ATP 2′(3′)-O(6-N′,N′-dimethylaminonaphthalenesulfonyl)ATP Cr(H20)4ATP, β,γ bidentate complex of chrom(III) tetraaquo-ATP Co(NH3)4ATP, β,γ bidentate complex of cobalt(III) tetraamino-ATP DANS-tryptophan 3′(2′)O-methylanthraniloyl-ATP 3′(2′)O(5-fluor-2,4-dinitro-phenyl)ATP 1,N 6-ethenoadenosine 5′-triphosphate 2′(3′)O-anthracenesulfonyl-ATP 2′(3′)O-pyrene-sulfonyl-ATP fluorescein isothiocyanate. Active Na+/K+-transport through mammalian cell membranes catalyzed by the sodium pump needs the interaction of high and low affinity ATP binding sites during catalysis (1Thoenges D. Schoner W. J. Biol. Chem. 1997; 272: 16315-16321Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). During pumping, a high affinity ATP site (E1ATP site)1 is phosphorylated when Na+/K+-ATPase (EC 3.6.1.37) is in its Na+-exporting E1 conformational state. Dephosphorylation, however, turns the enzyme to the K+-importing E2 conformation that binds ATP with low affinity (E2ATP site) (for a review, see Ref. 2Glynn I.M. Martonosi A.N. 2nd Ed. The Enzymes of Biological Membranes. 3. Plenum Publishing Corp., New York1985: 35-144Google Scholar). The kinetics of substrate hydrolysis of the enzyme vary with the nature of the nucleoside triphosphate. Although ATP hydrolysis proceeds in a negative cooperative way (3Neufeld A.H. Levy M.L. J. Biol. Chem. 1969; 223: 6493-6497Abstract Full Text PDF Google Scholar), inhibition of ATP hydrolysis by 2′,3′-O(2,4,6-trinitrophenyl)-ATP (TNP-ATP), a substance that is not hydrolyzed, was reported to be partially competitive and noncompetitive (4Moczydlowski E.G. Fortes P.A.G. J. Biol. Chem. 1981; 256: 2357-2366Abstract Full Text PDF PubMed Google Scholar). Moreover, 2′(3′)-O(6-N′,N′-dimethylaminonaphthalenesulfonyl)-ATP (DANS-ATP) and 8-N3-DANS-ATP, which are not hydrolyzed either, show a positive cooperative effect during interaction with Na+/K+-ATPase (1Thoenges D. Schoner W. J. Biol. Chem. 1997; 272: 16315-16321Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). MgATP complex analogs can discriminate between E1ATP and E2ATP binding sites (5Schoner W. Thönges D. Hamer E. Antolovic R. Buxbaum E. Willeke M. Serpersu E.H. Scheiner-Bobis G. Bamberg E. Schoner W. The Sodium Pump: Structure, Mechanism, Hormonal Control, and Its Role in Disease. Steinkopff Verlag, Darmstadt, Germany1994: 332-341Crossref Google Scholar). Although Cr(H20)4ATP (Cr-ATP) inactivates the E1ATP binding site, Co(NH3)4ATP (Co-ATP) inactivates the E2ATP site (6Pauls H. Bredenbröcker B. Schoner W. Eur. J. Biochem. 1980; 109: 523-533Crossref PubMed Scopus (42) Google Scholar, 7Linnertz H. Thönges D. Schoner W. Eur. J. Biochem. 1995; 232: 420-424Crossref PubMed Scopus (27) Google Scholar, 8Scheiner-Bobis G. Fahlbusch K. Schoner W. Eur. J. Biochem. 1987; 168: 123-131Crossref PubMed Scopus (35) Google Scholar). The ribosyl-modified TNP-ATP is known as a substance that binds in relation to ATP with increased affinities to both ATP binding sites (4Moczydlowski E.G. Fortes P.A.G. J. Biol. Chem. 1981; 256: 2357-2366Abstract Full Text PDF PubMed Google Scholar, 9Scheiner-Bobis G. Antonipillai J. Farley R.A. Biochemistry. 1993; 32: 9592-9599Crossref PubMed Scopus (34) Google Scholar). Furthermore, we showed recently that ribosyl-modified DANS-ATP binds with much higher affinity to the E2ATP site than to the E1ATP site (1Thoenges D. Schoner W. J. Biol. Chem. 1997; 272: 16315-16321Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). This peculiar phenomenon is not understood very well. A better understanding would be helpful not only to find more protein-reactive ATP derivatives with a preference for the low affinity E2ATP binding site but also to realize whether the method of analysis of the complex kinetics with a Koshland-Némethy-Filmer model of two cooperating ATP sites is generally applicable to all ATP derivatives. Hence, such a model would describe a general property of the enzyme. This would also include that it is justified to extrapolate from the knowledge of microscopic dissociation constants of the E1ATP and E2ATP sites, obtained from the inactivation with MgATP complex analogs (5Schoner W. Thönges D. Hamer E. Antolovic R. Buxbaum E. Willeke M. Serpersu E.H. Scheiner-Bobis G. Bamberg E. Schoner W. The Sodium Pump: Structure, Mechanism, Hormonal Control, and Its Role in Disease. Steinkopff Verlag, Darmstadt, Germany1994: 332-341Crossref Google Scholar), to the complex macroscopic kinetics of Na+/K+-ATPase (1Thoenges D. Schoner W. J. Biol. Chem. 1997; 272: 16315-16321Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Therefore, we started a careful kinetic analysis of a number of ATP and nucleoside analogs with modified ribose and polyphosphate moieties. Analysis of all of the substances for their microscopic dissociation constants of the E1ATP and E2ATP sites by previously reported methods (1Thoenges D. Schoner W. J. Biol. Chem. 1997; 272: 16315-16321Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 5Schoner W. Thönges D. Hamer E. Antolovic R. Buxbaum E. Willeke M. Serpersu E.H. Scheiner-Bobis G. Bamberg E. Schoner W. The Sodium Pump: Structure, Mechanism, Hormonal Control, and Its Role in Disease. Steinkopff Verlag, Darmstadt, Germany1994: 332-341Crossref Google Scholar) and of the kinetics of overall hydrolysis or substrate inhibition by use of a model of two interacting ATP sites revealed that the previously published Koshland-Némethy-Filmer model describes sufficiently well all kinetics. The correlation of kinetic data with structural data led to a postulate of minimal requirements of ATP analogs for high affinity interaction with the low affinity E2ATP site. Such properties are a "thickened" adenine ring because of stacking of a ribose-ligated bulky fluorophore at an flexible ribose moiety. 1-Pyrenesulfonyl chloride, 1-anthracenesulfonyl chloride, and N-methylisatoic anhydride were purchased from Molecular Probes (Eugene, OR). DANS-tryptophan (DANS-TRP) was supplied by Serva (Heidelberg, Germany). Lab-Trol, a protein standard used in clinical chemical analysis, was delivered by Baxter Dade (Dudingen, Switzerland). Na+/K+-ATPase from pig kidneys with a specific activity of 18–25 units/mg was purified by a modification of Jørgensen′s procedure (10Jφrgensen P.L. Biochim. Biophys. Acta. 1974; 356: 36-52Crossref PubMed Scopus (773) Google Scholar) and measured by a coupled optical assay (1Thoenges D. Schoner W. J. Biol. Chem. 1997; 272: 16315-16321Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Protein was determined by the method of Lowry et al. (11Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using Lab-Trol as a standard. When the inhibitory effect of ATP analogs on Na+/K+-activated ATP hydrolysis was studied, variable concentrations of all nucleotides were included into the optical assay. For measurement of the hydrolysis of 3′(2′)-O-methylanthraniloyl-ATP (MANT-ATP) and 3′(2′)-O(5-fluor-2,4-dinitrophenyl)-ATP (FDNP-ATP), no ATP was added to the optical assay. The reaction was generally started with 0.1 units of Na+/K+-ATPase. The structures of ATP derivatives used in this study are shown in Fig. 1. 1-N 6-Ethenoadenosine 5′-triphosphate (ε-ATP) was obtained by a method of Barrio et al. (12Barrio J.R. Secrist III, J.A. Leonard N.J. Biochem. Biophys. Res. Commun. 1972; 46: 597-604Crossref PubMed Scopus (296) Google Scholar). MANT-ATP, MANT-cAMP, and TNP-ATP were prepared according to Hiratsuka and Uchida (13Hiratsuka T. Uchida K. Biochim. Biophys. Acta. 1973; 320: 635-647Crossref PubMed Scopus (143) Google Scholar) and Hiratsuka (14Hiratsuka T. Biochim. Biophys. Acta. 1983; 742: 496-508Crossref PubMed Scopus (396) Google Scholar). FDNP-ATP, 2′(3′)-O-pyrenesulfonyl-ATP (PYRS-ATP), 2′(3′)-O-Anthracenesulfonyl-ATP (ANTS-ATP) and dansylated nucleotides were synthesized by a modified method of Chuan (1Thoenges D. Schoner W. J. Biol. Chem. 1997; 272: 16315-16321Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 15Chuan H. Wang J.H. J. Biol. Chem. 1988; 263: 13003-13006Abstract Full Text PDF PubMed Google Scholar, 16Chuan H. Lin J. Wang J.H. J. Biol. Chem. 1989; 264: 7981-7988Abstract Full Text PDF PubMed Google Scholar). DANS-adenosine was obtained from DANS-AMP by treatment with alkaline phosphatase (17Chaconas G. van de Sande J.H. Methods Enzymol. 1980; 65: 75-85Crossref PubMed Scopus (191) Google Scholar). The purity of the compounds was controlled by thin-layer chromatography (Silica gel 60 F254, 10:6:3n-butyl alcohol/water/acetic acid) and by UV, fluorescence, and NMR spectroscopy. To determine the concentration of ATP analogs, the amount of ribose and phosphate was analyzed by the orcin test and the method of Fiske and Subarrow, respectively. Microscopic dissociation constants of the complexes of E1ATP and E2ATP binding sites with the ATP analogs of interest were estimated from the protective effect of the substances against the inactivation of Na+/K+-ATPase by Cr-ATP and Co-ATP (1Thoenges D. Schoner W. J. Biol. Chem. 1997; 272: 16315-16321Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Cr-ATP has been shown to inactivate by tight binding and by phosphorylation the E1ATP site of Na+/K+-ATPase (6Pauls H. Bredenbröcker B. Schoner W. Eur. J. Biochem. 1980; 109: 523-533Crossref PubMed Scopus (42) Google Scholar) and Co-ATP by forming a tight complex with the E2ATP site (8Scheiner-Bobis G. Fahlbusch K. Schoner W. Eur. J. Biochem. 1987; 168: 123-131Crossref PubMed Scopus (35) Google Scholar). 1 unit of Na+/K+-ATPase was incubated in a total volume of 250 μl at 37 °C in 60 mm imidazole, HCl, pH 7.25, and increasing concentrations of Cr-ATP (10–100 μm) or Co-ATP (100–1000 μm). The inactivation of Na+/K+-ATPase was recorded in the absence and presence of the respective ATP analog by transferring an aliquot of 20 μl of the reaction medium in intervals of 15 min to the optical assay. Rate constants of inactivation and dissociation constants of the enzyme·nucleotide complexes were determined by the method of Piszkiewics and Smith (18Piszkiewics D. Smith E.L. Biochemistry. 1971; 10: 4544-4552Crossref PubMed Scopus (44) Google Scholar) and the two-site model (see Equation 2). Na+/K+-ATPase was inactivated by FITC at pH 9 (9Scheiner-Bobis G. Antonipillai J. Farley R.A. Biochemistry. 1993; 32: 9592-9599Crossref PubMed Scopus (34) Google Scholar) and washed in 50 mmTris, HCl, pH 7.5. An amount of this enzyme equivalent to 0.1 units of untreated Na+/K+-ATPase or 0.1 units of native enzyme was assayed for K+-activated phosphatase. Increasing concentrations of p-nitrophenyl phosphate were incubated with 5 mm MgCl2 and 50 mm KCl in the presence or absence of variable amounts of ATP analogs on microtiter plates at room temperature. The reaction was stopped after 15 min of hydrolysis with 1 n NaOH. Absorbance was measured at 410 nm. A two-site model according to Koshland, Némethy, and Filmer (Fig. 2) was used to analyze the kinetics of ATP analogs (1Thoenges D. Schoner W. J. Biol. Chem. 1997; 272: 16315-16321Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 19Koshland Jr., D.E. Nemethy G. Filmer D. Biochemistry. 1966; 5: 365-385Crossref PubMed Scopus (2200) Google Scholar). The equations for the rate of hydrolysis of substrate S in presence of an inhibitor I (see Equation 1) or the rate of inactivation by an inhibitor I in presence of a protecting ligand S (see Equation 2) were derived according to Segel (20Segel E.H. Enzyme Kinetics. John Wiley & Sons, Inc., New York1975Google Scholar). The latter equation takes into account the specificity of Cr-ATP to the E1ATP binding site (y = 1, c against infinity) and the specificity of Co-ATP to the E2ATP binding site (y against infinity). All computations and calculations of binding parameters were performed by use of the program Prism 2.0 of GraphPad Software Inc., San Diego, CA 92121. The overall reaction in presence of an inhibitor is expressed as υpVp,max=[S]z×Kd+[S]2a×Kd21+2×[S]Kd+[S]2a×Kd2+2×[S]×[I]b×Kd×KI+2×[I]KI+[I]2c×KI2Equation 1 and inactivation by Cr-ATP or Co-ATP in the presence of a ligand is expressed as υiVi,max=[I]y×KI+[S]b×Kd×KI+[I]2c×KI21+2×[I]KI+[I]2c×K12+2×[S]Kd+2×[S]×[I]b×Kd×KI+[S]2a×Kd2Equation 2 After exchange of the removable protons, the 1H NMR spectra were recorded in D2O in a Bruker AM 400 MHz spectrograph. The coupling constants of the protons of ribose allowed an evaluation of the time-averaged structure (21Sänger W. Principles of Nucleic Acid Structures. Springer-Verlag New York Inc., New York1984Crossref Google Scholar, 22Olson W.K. Sussman J.L. J. Am. Chem. Soc. 1982; 104: 270-278Crossref Scopus (198) Google Scholar). ATP analogs are termed as flexible if 3 Hz < (J1,2 and J3,4) < 7 Hz and are termed as fixed if 3 Hz > (J1,2 or J3,4) > 7 Hz. Time-resolved fluorescence measurements were performed by the time-correlated single photon-counting method using synchrotron radiation as a source of the excitation light (23Obsil T. Merola F. Lewit-Bentley A. Amler E. FEBS Lett. 1998; 426: 297-300Crossref PubMed Scopus (12) Google Scholar). The instrumental function and fluorescence decays were measured sequentially during several 10 of cycles and stored in groups of 2048 channels each (time interval 44.2 ps/channel; total number of counts exceeded 106 for each measurement; temperature adjusted to 25 °C by water bath; excitation wavelength λ = 290 nm; emission wavelength λ = 415 nm; excitation and emission bandwidth 9 nm). The total fluorescence decays were collected with the excitation polarizer set to the vertical position and the emission polarizer set at 54.7° (magic angle). A total of 2–3 million counts were collected in each decay, and the maximum entropy method was used for data analysis. The final lifetime distribution was split into as many species as there are peaks separated by two well defined maxima. The first order averaged lifetime τ was then calculated as Σ ciτi. Errors on averaged lifetimes are based on estimates of the repeatability of the measurements. Molecular properties of ATP analogs were determined using the semi-empirical method AM1 (24Dewar M.J.S. Zoebisch E.G. Healy E.F. Stewart J.P. J. Am. Chem. Soc. 1985; 107: 3902-3909Crossref Scopus (15064) Google Scholar) implemented in the program MOPAC 7. The geometric optimization is characterized by a minimization of the force constants. To learn whether the knowledge of the microscopic dissociation constant of the enzyme complex with an ATP derivative at a specific ATP site may facilitate the kinetic analysis of the overall reaction according to the Koshland-Némethy-Filmer model, the interaction of TNP-ATP with Na+/K+-ATPase was evaluated. Affinities of TNP-ATP for the two substrate binding sites (Fig. 2) were determined from their protective effect against the inactivation by Cr-ATP and Co-ATP (Fig. 3). TheK d = 0.7 ± 0.3 μm anda = 300 ± 100 of TNP-ATP obtained by the fitting process (Equation 2) to the data analyzed with both inactivating MgATP analogs is in good agreement with direct measurements in the native enzyme (4Moczydlowski E.G. Fortes P.A.G. J. Biol. Chem. 1981; 256: 2357-2366Abstract Full Text PDF PubMed Google Scholar). Furthermore, the analysis of inhibition of TNP-ATP on the hydrolysis of p-nitrophenyl phosphate withK I = 160 ± 20 μm in a FITC-treated enzyme but with K I = 7 ± 2 μm in native enzyme is indicative for negative cooperativity (Fig. 4). ATP hydrolysis by Na+/K+-ATPase also shows negative cooperativity (3Neufeld A.H. Levy M.L. J. Biol. Chem. 1969; 223: 6493-6497Abstract Full Text PDF Google Scholar). The complex kinetics of ATP hydrolysis in the presence of TNP-ATP could not be described quantitatively so far (4Moczydlowski E.G. Fortes P.A.G. J. Biol. Chem. 1981; 256: 2357-2366Abstract Full Text PDF PubMed Google Scholar). When the two-site model (Fig. 2, Equation 1) was applied to fit curves to experimental points, an excellent fit was obtained (Fig.5). Using the KI values of TNP-ATP as determined above for both ATP sites as microscopic dissociation constants, the experimental finding of downward-bending lines in the double reciprocal plot was quantitatively described by the parameters K d(ATP) = 0.3 ± 0.1 μm, K I(TNP-ATP) = 0.1 ± 0.05 μm, a = 400 ± 100,b = 70 ± 30, c = 2000 ± 500, and z = 8 ± 4 (Fig. 5). Hence, the knowledge of microscopic dissociation constants for the two ATP binding sites facilitates the fitting of kinetic data of the overall reaction, considerably. Because this procedure was already applied successfully in a previous study to describe the interaction of another ATP derivative with Na+/K+-ATPase (1Thoenges D. Schoner W. J. Biol. Chem. 1997; 272: 16315-16321Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), this procedure seems to be valid generally. Therefore, it was applied in the subsequent study as well.Figure 4Analysis of K+-activated hydrolysis of p-nitrophenylphosphatase in the presence of TNP-ATP. The effect of various concentrations of TNP-ATP on the activity of K+-activatedp-nitrophenylphosphatase was measured in native (top) and FITC-treated Na+/K+-ATPase (bottom). Treatment of Na+/K+-ATPase by FITC is known to block the Cr-ATP-sensitive E1ATP binding site but not the Co-ATP sensitive E2ATP binding site (7Linnertz H. Thönges D. Schoner W. Eur. J. Biochem. 1995; 232: 420-424Crossref PubMed Scopus (27) Google Scholar). The following concentrations of TNP-ATP were included into the phosphatase assay for native enzyme: ○, 10 μm; ▴, 20 μm; ■, 40 μm; ▪, no TNP-ATP; and for FITC-treated enzyme: ○, 25 μm; ▴, 75 μm; ■, 125 μm; ▪, no TNP-ATP. One typical experiment is shown. Insets, replot of the apparent affinities of potassium phosphatase for p-nitrophenyl phosphate against the TNP-ATP concentration. The K d (TNP-ATP) = 7 ± 2 μm in native enzyme and the K d(TNP-ATP) = 160 ± 20 μm in FITC-treated enzyme were extrapolated from the intercept of the straight line with theabscissa. U, units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Analysis of Na+/K+-activated hydrolysis of ATP in the presence of TNP-ATP. The influence of TNP-ATP on the overall reaction was studied by the optical assay. The following concentrations of TNP-ATP were included into the optical assay at variable concentrations of ATP: ○, 1 μm; ▴, 3 μm; ■, 5 μm; •, 7 μm;▪, no TNP-ATP. The solid lines are the result of an analysis according to the Koshland-Némethy-Filmer model (Equation1) using the parameters of Fig. 3 for the fitting process. The following parameters (S.D. of three different measurements) were obtained: K d(ATP) = 0.3 ± 0.1 μm, KI(TNP-ATP) = 0.1 ± 0.05 μm, a = 400 ± 100,b = 70 ± 30, c = 2000 ± 500, z = 8 ± 4. The ordinate was normalized to maximal velocity (V max/v). If not indicated,error bars are smaller than the symbols used.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In contrast to TNP-ATP and DANS-ATP (1Thoenges D. Schoner W. J. Biol. Chem. 1997; 272: 16315-16321Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 4Moczydlowski E.G. Fortes P.A.G. J. Biol. Chem. 1981; 256: 2357-2366Abstract Full Text PDF PubMed Google Scholar) other ribose-modified ATP analogs like MANT-ATP and FDNP-ATP are substrates of Na+/K+-ATPase (14Hiratsuka T. Biochim. Biophys. Acta. 1983; 742: 496-508Crossref PubMed Scopus (396) Google Scholar) (TableI). A careful analysis of the E1ATP and E2ATP sites, as described above, showed for FDNP-ATP identical affinities for both substrate binding sites (K d = 10 μm) and, hence, a Michaelis-Menten type of hydrolysis (data not shown). MANT-ATP, however, exhibited a positive cooperativity with aK d = 200 ± 50 μm for the E1ATP site and a K d = 80 ± 40 μm for the E2ATP site. This result could be corroborated by an analysis of the effect of MANT-ATP on K+-activated p-nitrophenylphosphatase in a E1ATP site-blocked enzyme. It gave for MANT-ATP theK I = 100 ± 20 μm in a FITC-treated enzyme and K I = 300 ± 50 μm in an untreated enzyme (data not shown). Apparently, modification of the E1ATP binding site by the adenine-imitating FITC (to which MANT-ATP binds with low affinity) enhances in a positive cooperative way binding of MANT-ATP at the E2ATP site. When the overall kinetics of substrate hydrolysis were analyzed with the Koshland-Némethy-Filmer model (Equation 1), the following parameters were obtained by the fitting process: for MANT-ATP, K d = 200 ± 50 μm, a = 0.4 ± 0.2,z = 0.3 ± 0.2; for FDNP-ATP,K d = 5 ± 2 μm, a= 1 ± 0.3, z = 1 ± 0.5; and for ATP,K d = 1 ± 0.2 μm,a = 100 ± 20, z = 15 ± 5 (Fig. 6). Hence, the individual ATP derivatives do not only differ in their individual affinities for the two ATP bindings sites (Table I) but also in the turnover rate (z value). The maximal velocity of Na+/K+-supported hydrolysis of ATP (V max = 30 units/ml) is about 15 times faster than that of FDNP-ATP and about 50 times faster than that of MANT-ATP.Table IKinetic parameters of ATP analogsATP analogFunctionE1ATPE2ATPμmμmATPSubstrate0.3 ± 0.1120.0 ± 30TNP-ATPInhibitor0.7 ± 0.3210 ± 70FDNP-ATPSubstrate10 ± 310 ± 3DANS-ATPInhibitor100 ± 100.3 ± 0.2ANTS-ATPInhibitor300 ± 1003 ± 1PYRS-ATPInhibitor400 ± 1004 ± 1MANT-ATPSubstrate200 ± 5080 ± 40GTPSubstrateNo effectNo effectDANS-GTPNo substrate, no inhibitorNo effect500 ± 200CTPSubstrateNo effectNo effectDANS-CTPNo substrate, no inhibitorNo effectNo effectAdenosineNo substrate, no inhibitorNo effectNo effectDANS-AdenosineInhibitorNo effect10 ± 3AMPNo substrate, no inhibitorNo effectNo effectDANS-AMPInhibitorNo effect1 ± 0.5c-AMPNo substrate, no inhibitorNo effectNo effectDANS-cAMPNo substrate, no inhibitorNo effectNo effectMANT-cAMPNo substrate, no inhibitorNo effectNo effectTRPNo substrate, no inhibitorNo effectNo effectDANS-TRPInhibitor50 ± 10No effectRibose-modified ATP analogs were analyzed for their respective microscopic affinities for the E1ATP and E2ATP sites from their protective effects against the inactivation of Na+/K+-ATPase by Cr-ATP or Co-ATP (see "Material and Methods"). "No effect" means no protection detectable against the inactivation. Open table in a new tab Ribose-modified ATP analogs were analyzed for their respective microscopic affinities for the E1ATP and E2ATP sites from their protective effects against the inactivation of Na+/K+-ATPase by Cr-ATP or Co-ATP (see "Material and Methods"). "No effect" means no protection detectable against the inactivation. The Hill coefficient is a measure of cooperativity. It is calculated as the first derivative of Equation 1 as log(v/(1 −v)) against log(S). For the Koshland-Némethy-Filmer model of two interacting substrate sites, this derivative is shown in Equation 3. When the above-evaluated data were used to calculate the change of cooperativity as a function of substrate concentration, Fig.7 resulted. It is well known from the work of Cornish-Bowden and Koshland (25Cornish-Bowden A. Koshland Jr., D.E. J. Mol. Biol. 1975; 95: 201-212Crossref PubMed Scopus (158) Google Scholar) that the cooperativity changes with the substrate concentration. Evidently negative cooperativity of ATP was most pronounced at very low ATP concentrations, i.e.at ATP concentrations that are commonly used for the demonstration of Na+-dependent phosphorylation (26Post R.L. Sen A.K. Rosenthal A.S. J. Biol. Chem. 1965; 240: 1437-1445Abstract Full Text PDF PubMed Google Scholar). Higher concentrations of ATP are known to affect the hydrolysis of the phospho intermediate (27Post R.L. Hegyvary C. Kume S. J. Biol. Chem. 1972; 247: 6530-6540Abstract Full Text PDF PubMed Google Scholar, 28Schwarzbaum P.J. Kaufman S.B. Rossi R.C. Garrahan P.J. Biochim. Biophys. Acta. 1995; 1233: 33-40Crossref PubMed Scopus (28) Google Scholar). nH=1z+2[S]aKd+2[S]2aKd2+2[S]2[I]abKd2KI+2[S][I]2acKdKI2+4[S][I]aKdKI+2[I]zKI+[I]2czKI2−[S]2azKd21z+[S]aKd+2[S]zKd+2[S]2aKd2+2[S][I]bzKdKI+4[S][I]abKd2KI+2[I]zKI+2[S][I]aKdKI+[I]2czK12+[S][I]2acKdKI2−[S]z2Kd−[S]azKd2Equation 3 It is unclear why the ribosyl-modified 2′(3′)-O-DANS-ATP and 3′(2′)-O-MANT-ATP bind with higher affinity to the E2ATP site than to the E1ATP site (1Thoenges D. Schoner W. J. Biol. Chem. 1997; 272: 16315-16321Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) and why 2′,3′-O-TNP-ATP preferably interacts with the E1ATP site (Table I). Moreover, it is unclear why TNP-ATP and DANS-ATP as ribosyl-modified ATP derivatives are not substrates, but inhibitors, and why other ribosyl-modified ATP derivatives like MANT-ATP and FDNP-ATP are hydrolyzed. A possible answer for the E2ATP site specificity is that a bulky fluorescent substituent at the ribose may achieve a better affinity for this site. In the case of oxidative phosphorylation, the particular behavior of DANS-ADP was explained by a hydrophobic (charge-transfer, stacking) complex between the adenine and dansyl moiety (29Schäfer G. Onur G. Eur. J. Biochem. 1979; 97: 415-424Crossref PubMed Scopus (36) Google Scholar). To test this hypothesis, a number of dansylated nucleotides were synthesized and studied on the overall reaction as well as for their protective effect against the inactivation of the E1ATP site by Cr-ATP and the E2ATP site by Co-ATP (Table I). Consistent with the above hypothesis, dansylated purine triphosphates like DANS-ATP and DANS-GTP showed a preference for the E2ATP site, but the pyrimidine derivative DANS-CTP did not. Because all dansylated derivatives of adenosine (DANS-ATP, DANS-AMP, and DANS-adenosine) except 2′-O-DANS-3′,5′-cyclic AMP (DANS-cAMP) showed a preferential binding to the E2ATP site, it was in fact possible that interaction of a bulky residue with the purine part is of importance for the E2ATP site specificity. To get additional information on the validity of this assumption, PYRS-ATP and ANTS-ATP were synthesized and investigated. In fact, ANTS-ATP and PYRS-ATP behaved like DANS-ATP (Table I). Because neither DANS-cAMP and MANT-cAMP nor dansylated tryptophan showed this specificity for the E2ATP site, it is evident that the interaction between the adenosine and the fluorophore per se is not responsible for the E2ATP site specificity but that, additionally, a characteristic pucker of the ribose moiety in the fluorescent nu

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