Tight Binding of Bulky Fluorescent Derivatives of Adenosine
to the Low Affinity E2ATP Site Leads to Inhibition of
Na+/K+-ATPase
ANALYSIS OF STRUCTURAL REQUIREMENTS OF FLUORESCENT ATP
DERIVATIVES WITH A KOSHLAND-NÉMETHY-FILMER MODEL OF
TWO INTERACTING ATP SITES*
Detlef
Thoenges
,
Evzen
Amler§,
Thomas
Eckert¶, and
Wilhelm
Schoner
From the
Institute of Biochemistry and Endocrinology
and ¶ Institute of Organic Chemistry,
Justus-Liebig-University Giessen, D-35392 Giessen, Germany and
§ Institute of Physiology, Czech Academy of Sciences,
Videnska 1083, Cz-142 20 Prague 4, Czech Republic
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ABSTRACT |
A Koshland-Némethy-Filmer model of two
cooperating ATP sites has previously been shown to explain the kinetics
of inhibition of Na+/K+-ATPase (EC
3.6.1.37) 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
(Kd = 0.7 µM) and to the
E2ATP site (Kd = 210 µM),
but 3'(2')-O-methylanthraniloyl-ATP in a positive
cooperative way with a lower affinity to the E1ATP binding
site (Kd = 200 µM) than to the
E2ATP binding site (Kd = 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 (Kd = 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.
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INTRODUCTION |
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 (1). 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. 2).
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 (3), 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 (4). 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 (1). MgATP
complex analogs can discriminate between E1ATP and
E2ATP binding sites (5). Although
Cr(H20)4ATP (Cr-ATP) inactivates the
E1ATP binding site, Co(NH3)4ATP
(Co-ATP) inactivates the E2ATP site (6-8). The
ribosyl-modified TNP-ATP is known as a substance that binds in relation
to ATP with increased affinities to both ATP binding sites (4, 9).
Furthermore, we showed recently that ribosyl-modified DANS-ATP binds
with much higher affinity to the E2ATP site than to the
E1ATP site (1). 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 (5),
to the complex macroscopic kinetics of
Na+/K+-ATPase (1).
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 (1, 5) 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.
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MATERIALS AND METHODS |
Chemicals--
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).
Enzyme and Assays--
Na+/K+-ATPase
from pig kidneys with a specific activity of 18-25 units/mg was
purified by a modification of Jørgensen's procedure (10) and measured
by a coupled optical assay (1). Protein was determined by the method of
Lowry et al. (11) 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.
Synthesis of ATP Analogs--
The structures of ATP derivatives
used in this study are shown in Fig. 1.
1-N6-Ethenoadenosine 5'-triphosphate (
-ATP)
was obtained by a method of Barrio et al. (12). MANT-ATP,
MANT-cAMP, and TNP-ATP were prepared according to Hiratsuka and Uchida
(13) and Hiratsuka (14). 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 (1, 15, 16).
DANS-adenosine was obtained from DANS-AMP by treatment with alkaline
phosphatase (17). The purity of the compounds was controlled by
thin-layer chromatography (Silica gel 60 F254, 10:6:3
n-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.

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Fig. 1.
Structures of ATP analogs. The ribose of
various ATP analogs exhibits different twisted and envelope
conformations. In 3'-O-MANT-ATP, the ribose is shown in the
C2'endo conformation. In 2'-O-DANS-cAMP, the
ribose is fixed in the C3'endo conformation by the cyclic
phosphate. The difference between 2',3'-O-TNP-ATP and
3'-O-FDNP-ATP is mainly the fixation of the ribose in the
O4'endo conformation by a Meisenheimer
complex.
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Determination of the Microscopic Dissociation Constant of an ATP
Analog to a Specific ATP Binding Site of Na+/K+
ATPase from Its Protective Effect against the Inactivation by Cr-ATP or
Co-ATP--
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 (1). Cr-ATP
has been shown to inactivate by tight binding and by phosphorylation
the E1ATP site of Na+/K+-ATPase (6)
and Co-ATP by forming a tight complex with the E2ATP site
(8). 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 (18) and the two-site model
(see Equation 2).
Influence of ATP Analogs on the Activity of
K+-activated p-Nitrophenylphosphatase in Native or
FITC-treated
Na+/K+-ATPase+--
Na+/K+-ATPase
was inactivated by FITC at pH 9 (9) and washed in 50 mM
Tris, 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.
Kinetic Evaluation of a Two-site Competitive Model of Koshland,
Némethy, and Filmer--
A two-site model according to Koshland,
Némethy, and Filmer (Fig. 2) was
used to analyze the kinetics of ATP analogs (1, 19). 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 (20). 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.

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Fig. 2.
General allosteric model of hydrolysis and
competitive inhibition at two binding sites. The hydrolysis and
competitive inhibition at two binding sites was described by a
Koshland-Némethy-Filmer model (1,19). The dissociation constants
of the substrate complex Kd and the inhibitory
complex KI at the first binding site are altered
by the interaction factors a, b, and c
for further binding at the second site. The rate constant of product
formation, kp, is influenced by a factor
z when product is formed by the double-occupied SES complex.
The rate constant of inhibitor formation ki is
influenced by a factor y when inhibitor is formed by the
double-occupied IEI complex. E = free enzyme;
S = substrate; I = inhibitor;
p = product, E*I = inactive
enzyme.
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The overall reaction in presence of an inhibitor is expressed as
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(Eq. 1)
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and inactivation by Cr-ATP or Co-ATP in the presence of a ligand
is expressed as
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(Eq. 2)
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Determination of Structural Factors in ATP Analogs--
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 (21, 22). 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 (23). 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
= 290 nm; emission wavelength
= 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
was then calculated as
ci
i. 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 (24) implemented in the program MOPAC 7. The
geometric optimization is characterized by a minimization of the force constants.
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RESULTS |
Analysis of the Interaction of TNP-ATP with High and Low Affinity
ATP Binding Sites of Na+/K+ATPase--
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). The
Kd = 0.7 ± 0.3 µM and
a = 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 (4). Furthermore, the analysis of inhibition of TNP-ATP on the
hydrolysis of p-nitrophenyl phosphate with
KI = 160 ± 20 µM in a
FITC-treated enzyme but with KI = 7 ± 2 µM in native enzyme is indicative for negative
cooperativity (Fig. 4). ATP hydrolysis by
Na+/K+-ATPase also shows negative cooperativity
(3). The complex kinetics of ATP hydrolysis in the presence of TNP-ATP
could not be described quantitatively so far (4). 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 Kd(ATP) = 0.3 ± 0.1 µM, KI(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 (1), this
procedure seems to be valid generally. Therefore, it was applied in the
subsequent study as well.

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Fig. 3.
Determination of the affinities of TNP-ATP
for the ATP sites of Na+/K+-ATPase. The
affinities of TNP-ATP for the ATP sites were determined from the effect
against the inactivation by Cr-ATP (top) and Co-ATP
(bottom). It is noteworthy that TNP-ATP protects against the
inactivation by Cr-ATP and that it accelerates the inactivation by
Co-ATP at low concentrations of TNP-ATP. Both cases, however, are
described by the same microscopic dissociation constants. The following
TNP-ATP concentrations were included into the inactivation assay for
Cr-ATP: , 0.5 µM; , 1 µM; , 3 µM; , 5 µM; , no TNP-ATP; and for
Co-ATP: , 50 µM; , 100 µM; , 200 µM; , no TNP-ATP. The ordinates were normalized to the
maximal inactivation rate constant. One typical experiment is shown.
The lines and parameters are the result of fitting of the
data according to the Koshland-Némethy-Filmer model (Equation 2):
Kd(Co-ATP) = 0.6 ± 0.2 µM,
c = 500 ± 200, Kd(Cr-ATP) = 20 ± 3 µM, Kd(TNP-ATP) = 0.7 ± 0.3 µM, a = 300 ± 100.
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Fig. 4.
Analysis 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+-activated
p-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 (7). 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 Kd (TNP-ATP) = 7 ± 2 µM in native enzyme and the Kd
(TNP-ATP) = 160 ± 20 µM in FITC-treated enzyme were
extrapolated from the intercept of the straight line with the
abscissa. U, units.
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Fig. 5.
Analysis 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 (Equation 1) using the parameters of Fig. 3 for the fitting process. The
following parameters (S.D. of three different measurements) were
obtained: Kd(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
(Vmax/v). If not indicated,
error bars are smaller than the symbols used.
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Analysis of the Interaction of MANT-ATP and FDNP-ATP with High and
Low Affinity ATP Binding Sites of Na+/K+
ATPase--
In contrast to TNP-ATP and DANS-ATP (1, 4) other
ribose-modified ATP analogs like MANT-ATP and FDNP-ATP are substrates of Na+/K+-ATPase (14) (Table
I). A careful analysis of the
E1ATP and E2ATP sites, as described above,
showed for FDNP-ATP identical affinities for both substrate binding
sites (Kd = 10 µM) and, hence, a
Michaelis-Menten type of hydrolysis (data not shown). MANT-ATP,
however, exhibited a positive cooperativity with a
Kd = 200 ± 50 µM for the
E1ATP site and a Kd = 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 the
KI = 100 ± 20 µM in a
FITC-treated enzyme and KI = 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, Kd = 200 ± 50 µM, a = 0.4 ± 0.2, z = 0.3 ± 0.2; for FDNP-ATP, Kd = 5 ± 2 µM, a = 1 ± 0.3, z = 1 ± 0.5; and for ATP, Kd = 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
(Vmax = 30 units/ml) is about 15 times faster
than that of FDNP-ATP and about 50 times faster than that of
MANT-ATP.
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Table I
Kinetic parameters of ATP analogs
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.
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Fig. 6.
Analysis of the
Na+/K+-activated hydrolysis of ATP and
MANT-ATP. The hydrolysis of MANT-ATP by
Na+/K+-ATPase was compared with the hydrolysis
of ATP. Various concentrations of both substrates were included into
the optical assay (see "Materials and Methods"). ATP ( ) shows
the known phenomenon of negative cooperativity
(Kd(ATP) = 1 ± 0.2 µM,
a = 100 ± 20, z = 15 ± 5),
whereas MANT-ATP ( ) is hydrolyzed with positive cooperativity
(Kd(MANT-ATP) = 200 ± 50 µM,
a = 0.4 ± 0.2, z = 0.3 ± 0.2). The affinities were determined by the use of the two-site model
(Equation 2, I = 0). Initial values for the fitting process were
taken from the protective effect against metal-ATP analogs (Table I).
Symbols represent the S.D. of three different measurements.
The ordinate was normalized to maximal velocity
(v/Vmax). If not indicated,
error bars are smaller than the symbols used.
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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 (25) 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 (26). Higher
concentrations of ATP are known to affect the hydrolysis of the phospho
intermediate (27, 28).
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(Eq. 3)
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Fig. 7.
Simulation of the Hill coefficient according
to the kinetic parameters of the two-site model. The kinetic
parameters of ATP, MANT-ATP, and FDNP-ATP were used to calculate the
Hill coefficient in dependence on the substrate concentration (Equation 3). ATP shows large negative cooperativity at low concentrations, which
is determined by the high affinity to the E1ATP site and
the low affinity to the E2ATP site. This effect is lowered
by the accelerating effect of ATP onto the rate constant of the
double-occupied enzyme complex at higher concentrations. In contrast,
MANT-ATP exhibits positive cooperativity in its binding affinities, but
this effect is lowered by the decelerating influence on the rate
constant of the double-occupied enzyme complex. FDNP-ATP, however,
shows no cooperative effect on the binding affinities and the rate
constant of hydrolysis.
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Analysis of the E2ATP Site Specificity of
Ribose-modified Fluorescent ATP Derivatives--
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 (1) 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 (29). 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
nucleoside is needed to achieve a binding at the E2ATP
site. In this context 3',5'-cyclo-AMP is known to exhibit a stabile
C3'endo conformation of its ribose because of the
intramolecular 3',5' phosphodiester bond (21). TNP-ATP, furthermore,
may have a stable conformation of its ribose because of the formation
of a Meisenheimer complex of the trinitrophenyl residue with the 2' and
3' hydroxyl groups (Fig. 1). In other derivatives, however, the ribose
shows several conformations because its pucker is not restricted by the
attached fluorophore (21).
1H NMR Studies and Dynamic Fluorescent Measurements of
ATP Analogs--
The above findings seem to indicate that the nature
and conformational flexibility or stability of a given ATP derivative is of importance for its ATP site specificity and also for the question
of whether a given ATP analog is a substrate or an inhibitor. The
specificity for the binding sites in turn determines the degree of
interaction between ATP sites. To get more reliable and independent data on the structure and conformational dynamics of the ribose moiety
of some important ATP derivatives, we analyzed them by 1H
NMR and dynamic fluorescence spectroscopy. 1H NMR
measurements in D2O revealed changes of the coupling
constants of substituted ribose atoms C1'-4' because of
attached fluorescent residues (Table II).
Analysis of the data according to the concept of pseudorotation (21,
30) showed that ribose in ATP, MANT-ATP, FDNP-ATP, and DANS-ATP is
flexible and oscillates between the two major conformations,
C2'endo and C3'endo (21) (Fig. 1). TNP-ATP and
DANS-cAMP, however, had a fixed ribose in a specific conformation
because of the 2',3'-O-trinitrophenyl group of the
Meisenheimer complex and the bridge of the 3',5'-diphosphoester, respectively (Table II). Unfortunately, it was not possible to calculate a specific pucker from the 1H NMR data.
Restricted dynamics of puckering of ribose by 2' and 3'-O-ribosyl substitutes, however, may explain the
preference of dansylated purine derivatives for the E2ATP
site (Table I). Provided stacking between fluorophores and the adenine
ring takes place (29), this should be demonstrable as energy transfer
between a fluorescent adenine moiety (i.e.
1,N6-etheno-adenine,
-adenine) and the DANS
residue. Evidently, a decrease of the fluorescence lifetime of
-adenine by a DANS residue attached to the ribose exists (Table
III). The efficiency of energy transfer
was much more pronounced in the dansylated
-cAMP than in the
dansylated
-ATP. Apparently both fluorophores are closer in the cAMP
derivative than in the ATP derivative.
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Table II
Coupling constants of the ribose of various ATP derivatives
The 1H NMR spectra of ATP analogs were recorded in a 400-MHz
Bruker spectrograph in D2O at room temperature. The
conformational state was calculated from the coupling constants (J)
according to Olson and Sussman (22) and Altona and
Sundaralingham (30).
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Table III
Fluorescence lifetimes of the etheno group in dansylated ATP analogs
The averaged fluorescence lifetime of the etheno group was estimated
with and without a dansylated substituent in -ATP and -cAMP. A
change in the lifetimes is indicative for the existence of a
charge-transfer (stacking) complex between the dansyl and adenine
moiety. For experimental parameters, see "Materials and Methods."
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Molecular Modeling of TNP-ATP, DANS-ATP, and DANS-cAMP--
To get
additional information and to visualize differences in the structures
of TNP-ATP, DANS-ATP, and DANS-cAMP, we also performed molecular
modeling of these molecules (Fig. 8). The molecules were drawn according to the information from the
1H NMR and fluorescence spectra and were subjected to a
geometric optimization. Because calculation was done in vacuum and
because of the missing influence of the enzyme protein on the
conformation of the ribose, the structures obtained are only a first
approximation.

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Fig. 8.
Structures of TNP-ATP, DANS-cAMP, and
DANS-ATP. The structures were simulated according to the
structural information obtained by 1H NMR and fluorescence
spectroscopy. The ribose was drawn in the envelope conformation
O4'endo for TNP-ATP (top), in the
C3'endo conformation for DANS-cAMP (middle), and
in the C2'endo conformation for DANS-ATP
(bottom). The dansyl residue was orientated in proximity to
the adenine base. Energy minimization (see "Materials and Methods")
was performed to the structures to get information about whether the
conformational parameters of ATP analogs may correlate to the
corresponding binding affinities at the ATP sites of
Na+/K+-ATPase. Obviously, TNP-ATP exhibits
negative cooperativity (high affinity to the E1ATP site)
and a flat adenine moiety, whereas DANS-ATP exhibits positive
cooperativity (high affinity to the E2ATP site) and a
"bulky adenine." Probably, these conformations represent the
corresponding structures for high affinity binding to the individual
ATP sites.
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|
 |
DISCUSSION |
This study shows that a Koshland-Némethy-Filmer model of two
interacting ATP sites does not only describe quantitatively the
positive cooperative effects of DANS-ATP and DANS-8N3-ATP on Na+/K+-ATPase (1) but also the negative
cooperative effect of TNP-ATP on ATP hydrolysis (Fig. 5), which could
not be explained before (4). Moreover, the two-site model does not only
describe quantitatively complex kinetics of inhibitory effects but also
complex effects of substrate hydrolysis (Fig. 6) like those of ATP
(showing a negative allosteric effect), FDNP-ATP (showing a
Michaelis-Menten type of hydrolysis), or MANT-ATP (showing a positive
cooperative effect). Hence the two-site model of interacting ATP sites
describes quantitatively all types of interactions or induced fit in
Na+/K+-ATPase by any energy substrate or
inhibitor. As was shown by the example of TNP-ATP, fitting of the
experimental data to the two-site model (Fig. 2) was greatly
facilitated by the use of the microscopic dissociation constants for
the fitting process. These microscopic dissociation constants were
obtained from the protective effect of the analog against the
inactivating MgATP complex analogs Cr-ATP and Co-ATP (Fig. 3). As is
also shown in the case of TNP-ATP, quite similar data were obtained
when the affinities of TNP-ATP were taken from its inhibition of
K+-activated phosphatase in a FITC-treated
(E1ATP site-blocked) or native enzyme (Fig. 4). This
situation is consistent with the previous experience in the analysis of
the effects of DANS-ATP on Na+/K+-ATPase
(1).
The use of the model of interacting ATP sites and of microscopic
dissociation constants for its analysis allowed it to describe quantitatively the peculiar behavior of ATP and of its analogs to
induce all types of cooperativity in hydrolysis and inhibition of
Na+/K+-ATPase. Because the structural reasons
for the variation in cooperativity and in the specificity of substrate
sites were unclear but of considerable interest for the understanding
of the mechanism of the sodium pump, a more detailed analysis on the
interaction of Na+/K+-ATPase with a number of
ribosyl-modified fluorescent adenosine derivatives was carried out
(Tables 1-3). The comparison of the affinities of the ATP analogs for
the E1ATP and E2ATP sites (obtained from their
protective effects against the inactivation of
Na+/K+-ATPase by either Cr-ATP or Co-ATP)
revealed that substitution at the hydroxyl groups of the ribose in ATP
by bulky fluorescent substituents like dansyl, pyrenesulfonyl, or
anthracenesulfonyl residues led to a preferential binding to the
E2ATP site (Table I). An interaction with the
E2ATP site occurred also when the
-phosphate or
C5'-phosphate was missing, because DANS-AMP and
DANS-adenosine bound with high affinity to this site as well. But a
bulky residue alone does not seem to be sufficient to achieve high
affinity interaction with the E2ATP site, because DANS-GTP and DANS-CTP interfered only weakly with this site and DANS-TRP interacted with the E1ATP site (Table I). Apparently, there
is need of the adenine moiety. The fluorescent
2'-O-DANS-3',5'-cyclic-AMP also did not interfere at all
with any ATP site (Table I), although this compound showed the most
pronounced effect in energy transfer (and hence stacking) between the
adenine and the dansyl residue (Table III). Obviously, there are
additional structural parameters determining the E2ATP site
specificity of DANS-ATP, DANS-AMP, DANS-adenosine, ANTS-ATP, and
PYRS-ATP (Table I). The ribose conformation in those compounds is
certainly an important major additional determinant for the ATP site
specificity and cooperativity in Na+/K+-ATPase.
The concept of pseudorotation (30) allows description of the influence
of substituents on the structure of the ATP molecule. The ribose in
unsubstituted ATP exists in at least two preferred twisted
conformations (C2'endo and C3'endo) (21). There
is a low energy barrier between these conformations, and the atoms seem
to migrate (pseudorotate) around the ribose. The phosphate chain and
the adenine base are substituents of the ribose and can be interpreted
as two masses flickering on a puckered basis. A fluorescent substituent
of the ribose acts as a third mass and stabilizes a ribose conformation
by affecting the equilibrium between the various puckered ribose
conformations. It is able to stabilize a conformation and to prevent
the pseudorotation between twisted conformations (21). Information on
the flexibility of the ribose and on the extent of pseudorotation was
obtained by 1H NMR spectroscopy. The analysis of the
spectra and the coupling constants revealed that DANS-cAMP has a fixed
ribose (Table II). But a fixed ribose does not seem to decide whether
an ATP derivative interferes with a specific ATP site or not. TNP-ATP,
which has a fixed ribose conformation as well (Table II), shows a much
higher affinity for the E1ATP than for the
E2ATP site (Table I). Therefore, ribose puckering may
differ in both molecules. TNP-ATP, with its inflexible ribose moiety,
is not hydrolyzed by Na+/K+-ATPase (4). Hence
conformational flexibility of the ribose moiety seems of importance for
the catalytic process. Other ATP derivatives showing puckering of their
ribose despite of its substitution (Table II) are substrates of the
enzyme but with a much reduced turnover rate, namely MANT-ATP and
FDNP-ATP. They bind with high affinity to the E2ATP binding
site (Table I). The theory of transition states in enzyme catalysis
explains increasing affinities of a ligand to its sites by a greater
structural identity to the enzyme·substrate complex. This better
binding, however, is payed for by a decrease in the following
rate-determining steps (31, 32). It is probable, therefore, that ATP
analogs with a higher affinity to the E2ATP binding site
than the natural substrate ATP are much more slowly hydrolyzed by
Na+/K+-ATPase. Hence, DANS-ATP, showing a
flexible ribose but with a very high affinity for the E2ATP
site inducing the strongest positive cooperativity, is, unlike MANT-ATP
and FDNP-ATP, not a substrate of Na+/K+-ATPase.
The same allosteric effect may be employed to explain DANS-ATP
protection against Cr-ATP, because binding of DANS-ATP was shown to be
fully reversible (1) and binding of Cr-ATP to the E2ATP
site was not detectable at the low concentrations used in the
experiment (data not shown). ATP, FDNP-ATP, and MANT-ATP apparently
undergo changes in their ribose conformations during catalysis. They
seem to be able to switch their ribose pucker from a conformation that
binds to the E1ATP site (which in turn induce the
E2ATP binding site) to a conformation that dissociates from
the E2ATP binding site. Such a sequential inductive process is independent of the steric arrangement of the ATP sites on the enzyme
protein (33) and is consistent with the two-site model of Koshland,
Némethy, and Filmer (1). This means also that binding of ATP
analogs to the E2ATP site is only possible after binding to
the E1ATP binding site regardless of the question of the
localization of these ATP sites on one or more protein subunits (34).
Evidently, this is seen in the case of DANS-cAMP, DANS-TRP, DANS-GTP,
and DANS-CTP that cannot bind to the E1ATP site and, in
turn, not to the E2ATP site because of the change in the
phosphate region, the lack of ribose pucker, or the missing adenine moiety.
In summary the ATP binding sites in
Na+/K+-ATPase show all types of cooperativity
in their interaction with ATP analogs. A two-site model according to
Koshland, Némethy, and Filmer is able to explain the kinetic
behavior of all substrates and inhibitors of
Na+/K+-ATPase (Figs. 3-7). There is a high
degree of probability that within the adenosine moiety, the ribose
conformation in ATP analogs bears the information responsible for its
preferential affinity and cooperativity between the ATP sites of
Na+/K+-ATPase. Obviously, free pseudorotation
is necessary for the hydrolysis of ATP analogs, whereas restriction in
a specific ribose conformation leads to preferential binding to one of
the two ATP sites in Na+/K+-ATPase. A bulky
fluorescent mass at the 2'(3') position of the ribose that interacts
with the adenin moiety apparently supports high affinity binding at the
E2ATP site of Na+/K+-ATPase (Fig.
8). A simplified explanation for this finding is that more protein
substrate contacts exist within the E2ATP site with a bulky
substrate, which hence lead to a tighter binding of this more open ATP
site in its E2 conformation to a more voluminous ATP
molecule. The cavity of the ATP site in its E1
conformation, on the one hand, is too small to interact with such bulky
ATP analogs (35). The flexibility of ribose, on the other hand, allows
structural rearrangements of the ATP molecule and subsequently binding
to the E1ATP site with low affinity. This is a prerequisite for high affinity substrate binding at the E2ATP site
(according to the Koshland-Némethy-Filmer model of induced fit)
and for the induction of positive cooperativity. With this knowledge of structural substrate requirements of each of the ATP sites of Na+/K+-ATPase it should now be possible to
construct protein-reactive ATP derivatives that allow detection of the
amino acids forming the low affinity E2ATP site.
 |
ACKNOWLEDGEMENT |
We thank Dr. Fabienne Merola, LURE,
CNRS-MEN-CEA, Orsay, France and Dr. Otto Kalinowski, Institute of
Organic Chemistry, Giessen, Germany for their help with data analysis
and for using the equipment. We also thank W. Mertens for technical assistance.
 |
FOOTNOTES |
*
This work was supported by the Deutsche
Forschungsgemeinschaft through the Graduiertenkolleg
"Molekulare Biologie und Pharmakologie" Giessen, the
Volkswagen Foundation Hannover and the Fonds der Chemischen Industrie,
Frankfurt/M.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Institut für
Biochemie und Endokrinologie, Fachbereich Veterinärmedizin,
Justus-Liebig-Universität Giessen, Frankfurter Strasse 100, D-35392 Giessen, Federal Republic of Germany. Fax: +49 641 99 38179;
E-mail: Wilhelm.Schoner{at}vetmed.uni-giessen.de.
The abbreviations used are:
E1ATP
site, Cr(H20)4ATP-sensitive site with high
affinity for ATP; E2ATP site, Co(NH3)4ATP-sensitive site with low affinity
for ATP; TNP-ATP, 2',3'-O(2,4,6-trinitrophenyl)ATP; DANS-ATP, 2'(3')-O(6-N',N'-dimethylaminonaphthalenesulfonyl)ATP; Cr-ATP, Cr(H20)4ATP,
,
bidentate complex
of chrom(III) tetraaquo-ATP; Co-ATP, Co(NH3)4ATP,
,
bidentate complex of
cobalt(III) tetraamino-ATP; DANS-TRP, DANS-tryptophan; MANT-ATP, 3'(2')O-methylanthraniloyl-ATP; FDNP-ATP, 3'(2')O(5-fluor-2,4-dinitro-phenyl)ATP;
-ATP, 1,N6-ethenoadenosine 5'-triphosphate; ANTS-ATP, 2'(3')O-anthracenesulfonyl-ATP; PYRS-ATP, 2'(3')O-pyrene-sulfonyl-ATP; FITC, fluorescein isothiocyanate.
 |
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