Eur. J. Biochem. 104, 443-450 (1980)
Interaction of Fructose- 1,6-Bisphosphate Aldolase
with Adenine Nucleotides
Binding of 5’-Mononucleotides and Phosphates to Rabbit Muscle Aldolase
Andrzej A. KASPRZAK and Marian KOCHMAN
Division of Biochemistry, Institute of Organic and Physical Chemistry, Technical University of Wroclaw
(Received August 17, 1979)
Equilibrium dialyses, fluorescence quenching and kinetic measurements were used to characterize
binding of adenine mononucleotides to the rabbit muscle aldolase. Using equilibrium dialysis
method 3.37 tight and 3.88 weak binding sites for ATP per tetrameric enzyme molecule were found
with the respective Kd values equal to 0.024 and 0.45 mM. In the presence of fructose 1,6-bisphosphate ATP was bound only to the weak sites. Inactive aldolase covalently labelled at lysine-226
with glycerol-l-phosphate exhibited 3.16 binding sites for ATP with a Kd of 0.30 mM.
A blue shift and 90-fold increase of fluorescence intensity was observed upon binding 8-anilinel-naphthalenesulfonate (ansyl) to aldolase. It was demonstrated that at low concentration of ansyl
this fluorescence probe was predominantly bound to a hydrophobic part of aldolase active center,
and changes of the fluorescence of the bound dye can be used for calculation the binding parameters
of ligands to this enzyme. The dissociation constants for tight binding sites at pH 7.5 and 25”C,
measured by this method were as follows: adenosine 5’-tetraphosphate, 0.008 mM; ATP, 0.034 mM;
ADP, 0.30 mM; AMP, 3.70 mM; polytriphosphate, 0.025 mM; pyrophosphate, 0.046 mM; and
phosphate 0.78 mM. MgATP complex binds to aldolase with the affinity of at least two orders of
magnitude lower than free nucleotide.
Kinetic measurements revealed pure competitive inhibition of aldolase by ATP with a Ki of
0.04 mM. Several lines of evidence suggest that tight binding proceeds at the catalytic site of aldolase.
For these sites free energies of binding depend linearly on the net charge of interacting nucleotide.
Possible structural relationship of aldolase with other glycolytic enzymes is discussed.
The molecule of rabbit muscle aldolase consists
of four, nearly identical, subunits [l- 31. Each subunit has one active center at which two phosphate
binding sites were detected. One of them, Lys-107
accomodates the C-6 phosphate of fructose-1, ~ - P[4].
z
It has been suggested that the binding site for the C-1
phosphate group consists of Arg-148 [5] and Lys-146
[6]. Lys-227 forms a Schiff base intermediate with the
substrate. Blocking it results in the complete inactivaAbbreviations. Ansyl, 8-aniline-l-naphthalenesulfonate;
adenosine-5’-P4, adenosine 5’-tetraphosphate; CAMP, cyclic adenosine
3 ‘ : 5’-monophosphate; dihydroxyacetone-P, dihydroxyacetone
phosphate; fructose-1,6-Pz, D-fructose-1,6-bisphosphate;glyceraldehyde-3-P, glyceraldehyde 3-phosphate; Mops, 4-morpholine
propanesulfonate; pentanedio1-1,5-P~, pentanediol-1,5-bisphosphate; PPPi, polytriphosphate; Taps, N-tris-(hydroxymethy1)methyl3-aminopropanesulfonate.
Enzymes. Fructose-1,6-bisphosphatealdolase (EC 4.1.2.13);
hexokinase (EC 2.7.1.1); triosephosphate isomerase (EC 5.3.1.1);
glycerol-l-phosphate dehydrogenase (EC 1.1.1.8); phosphofructokinase (EC 2.7.1.11); phosphoglycerate kinase (EC 2.7.1.3); pyruvate kinase (EC 2.7.1.40).
tion of the enzyme [7]. The investigations on the
tertiary structure of aldolase are in progress [8] and
a plausible mechanism of catalysis was proposed [9].
However, relatively little is known about physiologically important effectors which may modulate
aldolase activity.
Spolter and coworkers [ 101 noticed that adenine
nucleotides are competitive inhibitors of aldolase
isozymes. ATP was found to be the most effective
inhibitor for muscle enzyme, while AMP was the
least effective one. The reverse order of inhibition was
observed for liver aldolase.
In the present paper the interaction of adenine
mononucleotides and phosphates with rabbit muscle
aldolase has been studied. It is shown that each subunit of this enzyme has one strong and one weak
binding site. Our study also demonstrated that affinity
of the tight site is much greater than it was previously
reported. The electronegative phosphate group of the
nucleotides plays a predominant role in the binding ;
a secondary role can be ascribed to adenosine moiety.
444
Some of the results presented here have been described
in a preliminary communication [ll].
MATERIALS AND METHODS
Reagents and Enzymes
Adenosine-5'-P4, ATP, ADP, CAMP, PPP;, PP;,
NADH, and fructose-l,6-Pz were obtained as sodium
salts from Sigma. Mops and Taps were purchased
from Calbiochem. Ansyl (analytical grade) and Visking
dialysis tubing were from Serva; adenosine was from
Reanal (Budapest, Hungary) ; dihydroxyacetone-P
(dimethyl-ketal) and ~~-glyceraldehyde-3-P
(diethylacetal) were from Sigma. Free trioses were prepared
following the manufacturer's instruction. All other
chemicals were purchased from POCh (Gliwice,
Poland).
Aldolase was prepared from fresh rabbit skeletal
muscle using phosphocellulose chromatography [12].
Specific activities of four different preparations used
in this study were in the range 14.6-17.1 U/mg
protein. Glycerol-1-phosphate labelled aldolase was
prepared as described elsewhere [13]. Its specific activity was 0.12 U/mg protein.
All solutions were prepared less than 12 h before
use and filtered prior to experiments through 0.45 pm
microporous filters (Amicon). Deionized, glass bidistilled water was used throughout.
The purity of the nucleotides was determined
chromatographically on poly(ethy1eneimine)-cellulose
sheets (Merck) using an aqueous solution of LiCl as
a solvent [14]. With the exception of ADP all nucleotides were found to be pure. ADP was contaminated
with ATP and AMP (approximately 2%). Ansyl was
treated with active charcoal (Norit A), recrystallized
three times from water and purified chromatographically as described recently [15]. Only one fluorescent
spot was visible when 0.1 mg of purified ansyl was
chromatographed on silica gel plates (Merck) using
chloroform/methanol/acetic acid as a solvent. Pentanediol-1,5-P2 was synthetized in our laboratory by
Ms B. Krotkiewska [16].
Concentrations of the reagents were determined
spectrometrically using the following absorption coefficients: rabbit muscle aldolase, 0.938 cm-' ml mg-'
at 280 nm [17]; ansyl, 6.24 mM-l cm-' at 351 nm
[18]; 5'-nucleotides, 15.4mM-' cm-' at 259nm [19].
CAMP, 14.65 mM-' cm-' at 258 nm [20]; adenosine,
14.9 mM-' cm-'
at 259.5 nm [21]; NADH,
6.22 mM-' cm-' at 340 nm [22]. Molar concentrations of aldolase were calculated assuming molecular
weight of 160000 [l]. Concentrations of Mg2+ ions
were determined from complexometric titrations [23]
with spectrophotometric detection at 540 nm. Concentrations of fructose-l,6-Pz, dihydroxyacetone-P,
Interaction of Aldolase with Mononucleotides
and glyceraldehyde-3- P were measured enzymatically [24].
Aldolase, stored as a crystalline suspension in 50 %
saturated ammonium sulfate, was centrifuged, then
dissolved slowly in a cold buffer and dialyzed exhaustively against three changes of 150-fold volume
of an appropriate buffer at 5 "C for 8- 12 h. Unless
otherwise stated 50 mM Mops buffer, pH 7.5, supplemented with 1 mM EDTA was used to perform all
measurements. The activity of aldolase was determined spectrophotometrically [25]. All measurements
were performed using ACTA M-VI spectrophotometer (Beckman).
Analytical Procedures
Equilibrium Dialyses. Equilibrium dialyses were
carried out in a dialyzer consisting of two matched
lucite plates with ten shallow chambers cut into each.
The membranes were cut out from Visking dialysis
tubing, which was boiled in 50:d ethanol, in water
containing EDTA, and soaked for at least 1 h in a
buffer. 0.25 ml of aldolase solution was placed in one
half-cell and 0.25 ml of ATP was added to the other
half-cell. The dialyzer was rotated at 6 rev./min at
5 1 "C. After 20-22 h samples were removed from
the ligand compartments and the concentrations of
free nucleotide were determined spectrophotometrically. Control experiments showed that the equilibrium
was attained in 7 h. ATP was adsorbed neither on the
membrane nor on the dialyzer walls and 98 - 102 %
of the nucleotide was always recovered. For experiments in the presence of fructose-1,6-P2 a small correction (0.8 %) for the absorbance of this compound
at 259 nm [26] was employed. The enzyme retained
100 % activity after 22 h incubation period with 0.2 3 mM of ATP. The number of moles of the ligand
per mole of the enzyme, r was calculated according
to the equation :
r = W I o - 2 [LIf)/[Pl
(1)
where [Ll0denotes starting concentration of the ligand,
[LIf the equilibrium concentration of free ligand, and
[PI the protein concentration.
Fluorescence Measurements. Fluorescence measurements were carried out with Perkin-Elmer MPF-44
fluorescence spectrometer. The instrument was modified to permit thermostatting the cuvette with Rhodamine B in the monitor light beam. The modification
increased the stability of the spectrofluorometer
readings when it was operated in Ratio mode. Fluorescence titrations were done according to the following procedure. 3 ml of aldolase solution were
placed in a thermostatted holder of the instrument
for 15 min, then 5-15 p1 of ansyl solution was added.
Using a Hamilton microsyringe and a PB-600 repeating dispenser, 5 - 15 p1 aliquots of a ligand were
A. A. Kasprzak and M. Kochman
445
added. The content of the cuvette was stirred gently
with a small glass bar for 1 min, and the intensity of the
fluorescence was read out from a digital voltometer.
The samples were illuminated only for the time required to perform measurements. Excitation and emission wavelength were 350 and 485 nm, respectively.
Measurements were performed at 25 0.2"C or at
S k 1 "C. When the experiments were carried out at
5 "C, dry nitrogen was circulated through the sample
compartment to prevent water condensation. Changes
in fluorescence were corrected for dilution by the
added ligand and for the fluorescence of unbound
ansyl.
Kinetic Measurements. Steady-state kinetic measurements were obtained from initial velocities by
spectrophotometric recording the disappearance of
NADH, as described earlier [27]. Since sulfate anions
exert an inhibitory effect on aldolase, a lyophilized,
sulfate-free mixture of coupling enzymes : triosephosphate isomerase and glycerol-1-phosphate dehydrogenase (Sigma, type X) was employed. Assay velocities
were unaltered by increasing concentration of auxilliary enzymes and were proportional to the concentration of aldolase in the cuvette at all concentrations of
the nucleotide tested. The reaction was initiated by
adding 0.4 pg of aldolase diluted in 0.2% of bovine
serum albumin (Sigma, fraction V). Incubation of
aldolase in the cuvette for 15 min and initiation the
reaction with fructose-l,6-P2 resulted in identical
velocities.
Data Analysis
The data obtained from equilibrium dialysis were
fitted to one of the following equations:
(3)
where r is number of moles of ATP bound per mole
of aldolase, [LIf the free ATP concentration, Kd the
intrinsic dissociation constant for aldolase-ATP complex. A weighted, nonlinear least squares iterative
procedure [28] was employed. Statistical weights were
assumed to be proportional to the fraction of bound
ligand.
The binding parameters: n, Kd and AF,,, from
fluorescence titration were obtained by nonlinear least
squares fitting the experimental data points to Eqn (4) :
A F = AF,,,
-1
Kd
+ [Lit + n [PI
2 n [PI
p p K z i F p }(4)
2 n [PI
~
where APis the observed fluorescence change of ansyl
bound to aldolase, AF,,, is AF when the binding sites
are saturated with the ligand, Kd is the intrinsic dissociation constant, n the number of binding sites per
mole of the enzyme, p ] the total enzyme concentration,
and [L], the total ligand concentration. For the determination of Kd the enzyme concentration was kept
much lower than Kd, whereas opposite conditions
were used when number of binding sites was estimated.
For weakly bound ligands (Kd > 0.3 mM) determination of n is unreliable using this method at manageable enzyme concentrations. In these cases Kd was
calculated assuming n = 4, although any value of n
between 2 and 8 resulted in insignificantly different
Kd values.
For fluorescence titrations of alsolase-bound ansyl
with ATP performed in the presence of magnesium,
concentrations of the free nucleotide was calculated
from Eqn (5):
[ATP]f
- -
-
z
+ I z 2 + 4 K&~TP([ATPI,
-~
-
2 KihgATP
[ATP]h)
~.
(5)
+
Khg~~p[Mg2+ l tKM,ATP([ATP]~
where z = 1
- [ATP]h). The indices t, f and b are used to mark
total, free, and bound species, respectively. Concentration of ATP bound to aldolase was estimated from
fluorescence data according to Eqn (6) :
[ATPIb = [PIS (AF/AFmax)
(6)
where [PIs is total concentration of aldolase subunits.
Eqn (5) was derived from the definition of stability
constant for MgATP and conservation equations for
ATP and Mg2+, assuming that binding of MgATP
to aldolase is negligible. Calculation of stability
constant for MgATP was done following a recently
published method [29]; for our experimental conditions Kh,ATp = 2.3 . lo4 M-'.
The steady-state kinetic data were fitted to rectangular hyperbola as described by Wilkinson [30].
All calculations were performed on ODRA-1300
computers (Mera-Elwro, Wrodaw, Poland).
RESULTS
Equilibrium Dialysis Measurements
Binding isotherms for the interaction of native
aldolase and its glycerol-1-phosphate adduct with
ATP obtained by equilibrium dialysis method at 5 C
are presented in Fig.1 as Scatchard plots. The isotherms obtained in SO mM Mops-Na buffer, 1 mM
EDTA, pH 7.5 for native aldolase displays a nonlinear Scatchard plot (Fig.1A) which may be described by a model of two classes of independent
binding sites. The binding parameters obtained from
nonlinear least-squares fit of the data to Eqn (3) are:
n1 = 3.37 f 1.47, Kdl = 0.024 k 0.010 mM, 122
= 3.88 & 1.90, Kd2 = 0.45 & 0.30 mM.
446
Interaction of Aldolase with Mononucleotides
0
2
4
r
6
8
0
1
2
3
4
3
4
r
.
I
<
0
0
1
2
r
3
4
0
1
2
r
Fig. 1 , Binding isotherms of ATP to aldolase and its glycerol-I-phosphate adduct ohtained from equilihrium dialysis. Solid lines were
computed using n and & values specified below. (A) Native aldolase at pH 7.5, enzyme concentration 0.1 mM, free ATP concentration
= 0.024 mM, nz = 3.88, Kd2 = 0.45 mM. (B) Native aldolase at pH 8.5, enzyme concentration
range 0.007-2.7 mM, Itl = 3.37,
0.1 mM, free ATP concentration range 0.016-3 mM, n = 3.84, Kd = 0.075 mM. (C) Native aldolase at pH 7.5 in the presence of 3.8 mM
fructose-1,6-P2; same conditions as in (A); n = 3.8, Kd = 1.1 mM. (D) Glycerol-1-phosphate adduct of aldolase at pH 7.5, enzyme
concentration 0.1 mM, free ATP concentration range 0.07- 1.9 mM, other conditions as in (A); n = 3.16, Kd = 0.30 mM
In 60 mM Taps-Na buffer, 1 mM EDTA, pH 8.5
simple binding isotherm was obtained for the binding
of ATP to the native aldolase (Fig. 1 B), and the best
fit of the data to Eqn (2) resulted in n = 3.84 0.89
and Kd = 0.075 k 0.050 mM.
At pH 7.5 in the presence of 3.8 mM fructosel,6-P2, the binding of ATP to the tight sites is not
observed (Fig. 1C) and Eqn (2) was used to calculate
binding parameters: n = 3.80 k 1.60 and Kd = 1.10
k 0.36 mM.
The binding isotherm of ATP to the glycerol-lphosphate adduct of aldolase at pH 7.5 revealed one
type of identical and independent sites with n = 3.16
0.33 and Kd = 0.30 k 0.08 mM (Fig. 1D).
Interaction of Ansyl with Aldoluse
In the presence of aldolase the fluorescence maximum of ansyl was shifted from 520 to 485nm and
a large increase in fluorescence efficiency occurred
(Fig. 2). The dissociation constant for aldolase-ansyl
complex found from the titration of the dye with the
enzyme, when [aldolase] % [ansyl]was 0.32mM (Fig. 3).
The fluorescence enhancement factor calculated from
this measurement was approximately 90. The titration
of aldolase with ansyl (not shown) in the concentration range 0-5 mM did not lead to the saturation
of binding sites on the protein, however. Thus besides
a set of relatively tight sites for ansyl, weak sites are
also present on the aldolase molecule.
Emission wavelength
(nm)
Fig. 2. Influence of' aldolase and its glycerol-I-phospkate adduct
on the jluorescerice spectrum of ansyl. Excitation wavelength was
Native aldolase, 11 pM and ansyl, 23 pM; (----)
350 nm. (-)
glycerol-2-phosphate derivative of aldolase, 11 pM and ansyl,
23 pM; (. . . . . . .) ansyl, 23 pM
Binding Stoichiometry
of Aldoluse Substrates Observed
by Fluorescence Quenching of Enzyme-Bound Ansyl
The substrates of aldolase : fructose-l,6-P2, dihydroxyacetone-P, and glyceraldehyde-3-P as well as
their analog-pentanediol-1 ,5-Pz decreased the fluorescence of enzyme-bound ansyl. The degree of
quenching was similar for all these compounds and
447
A. A. Kasprzak and M. Kochman
0
I
-0.2
0
I
I
I
0.2
0.4
Q6
0.8
1.0
4
5
-AF/F,
I
0.2 0.4 0.6 0.8 1.0
[Aldolase subunits]~,,, (mM)
Fig. 3. Fluorescence titration of ansyl with aldolase. Concentration
of ansyl was 7.4 pM. The solid line represents best fit for K d
= 0.32mM and dF,,,,,= 51.5. Under the same conditions the
fluorescence intensity of free ansyl (7.4 pM) was 0.58
was approximately equal to 80%. The number of
binding sites obtained from the titrations of enzymebound ansyl with fructose-I ,6-P2, dihydroxyacetone-P,
glyceraldehyde-3-P, and pentanediol-1,5-P2 was 4.30,
3.90, 5.70 and 4.33, respectively when measured at
pH 7.5 and 25°C. However, the number of binding
sites for fructose-l,6-Pz was 3.40 when the measurements were performed at 5 "C. The quenching of the
fluorescence by the substrates has indicated that
binding of ligands to the active center of aldolase can
be monitored by this method.
Binding Parameters
for Nucleotides and Phosphates Measured
by Fluorometric Titrations of Aldolase-Bound Amy1
Similarly to the substrates, adenine nucleotides and
phosphates quenched the fluorescence of ansyl-aldolase
complex. These changes in fluorescence were used for
the determination of Kd values and the number of
binding sites for these compounds. All these ligands
quenched at saturating concentrations 70 - 85 % of
the initial fluorescence of bound ansyl. In these experiments ansyl concentrations used were in the range
5 -20 pM which is much below the dissociation constant for the ansyl-enzyme complex (0.32 mM). To
demonstrate directly that there is no measurable
competition between the ligands tested and ansyl for
the binding sites on aldolase the dissociation constants
for ATP were measured at four different dye concentrations: 0.012, 0.025, 0.049 and 0.123 mM, respectively. The Kd values obtained were 0.0339, 0.0269,
0.03 17 and 0.0327 mM, respectively. Thus at [ansyl]
0.32 mM competitive effects between ansyl and the
ligands tested are negligible.
Typical fluorescence titration with ATP at pH 7.5
and 25 "C is shown in Fig. 4. Experimental variance
calculated for different numbers of binding sites
-+
"
0
1
2
r
3
Fig. 4. Titration of ansyl-aldolase complex with ATP. Concentrations
aldolase and ansyl were 38 pM and 7 pM, respectively. (A) Semilogarithmic plot showing relative changes of the fluorescence versus
total ATP concentration. Solid line represents best fit calculated for
n = 4.59, Kd = 0.0345 mM, and AF,,,,,/Fo = 67.9%. (B) Dependence of experimental variance on the number of binding sites assumed. (C) Scatchard plot of the data from Fig.4A
exhibited a minimum at n = 4.59 (Fig.4B). Scatchard
plot for the titration data revealed one class of independent binding sites with Kd = 0.023 k 0.008 mM.
There is a close agreement of this value with Kd for
tight binding sites obtained from equilibrium dialysis.
This suggests convincingly that the method of fluorescence quenching can furnish us with the 'true'
values of binding parameters for the ligands interacting with the active center of aldolase.
Using this technique the binding parameters for
adenosine-5'-P4, ADP, AMP, PPP,, PPi and P, were
determined at pH 7.5 and 25 "C, and are summarized
in Table 1. Bjerrum plots for the binding of nucleotides and phosphates and the corresponding Scatchard
plots are typical for identical and independent sites.
The effect of CAMPand adenosine on the fluorescence
of bound ansyl was also examined. The former compound did not exert discernible influence on the fluorescence of bound ansyl when present at concentrations up to 30 mM. Adenosine at concentration 1.9 mM
quenched 2 % of the initial fluorescence indicating a
weak interaction with aldolase.
Perusal of Table 1 may lead to the conclusion that
there is linear relationship between net charge of the
448
Interaction of Aldolase with Mononucleotides
Table 1. Binding parameters f o r interaction of adenine nucleotides with aldoluse at p H 7.5, 25 "C,and I = 0.035 M
Values of net charge at pH 7.5 were calculated from the Hendersson-Hasselblach equation using the following pK, values for secondary
phosphate: ATP, 6.97 [31]; ADP, 6.65 [31]; AMP, 6.4 [21]; P,, 7.2 [32]; PP,, pKa3 = 5.77, pKa4 = 8.20 [32], PPP,, pKa3 = 2.30, pKa4
= 6.26, and pK,s = 8.90 [33]. It was assumed that for adenosine-5'-P4 the pK, for the secondary phosphate is 7.3, i.e. is 0.3 units higher
than for ATP. Ado-5'-P4, adenosine 5'-tetraphosphate
Ligand
Number of
determinations
n
Kd
Ado-5'- P4
ATP
ADP
AMP
PPP,
pp,
pi
3
8
3
4
2
3
2
4.7 5 0.5
4.5 f 0.4
4"
4"
3.8 f 0.4
5.3 k 0.3
4
0.008
0.034
0.30
3.70
0.025
0.046
0.78
Net charge
mM
a
For weakly bound ligands it was assumed that n
=
AG;
kJ/mol
* 0.002
f 0.004
4.61
- 3.76
- 2.86
- 1.93
- 4.00
- 3.17
- 1.67
-
f 0.03
k 0.40
f 0.003
f 0.010
k 0.03
-
29.09
25.51
20.11
13.88
26.27
24.76
17.74
4.
- 30
45
-25
3 4.0
a
L
='
-20
e
.
2 3.5
t
0
-
7
25 -15
6
3.0
a
-lo,
-5 ,/
5.5
I
0'
0
I
-1
I
-2
-3
-4
Net charge at pH 7.5
I
5.0
-5
z
,
Fig. 5. Dependence of stundurdfree energy changes on the net charge
of interacting ligand. Ado-5'-P4, adenosine 5'-tetraphosphate
-
-
4.5
t
4
I
-
' 4.0
nucleotide at pH 7.5 and its standard free energy
change upon binding to aldolase (Fig. 5). This relation
may be expressed as
AG':
=
5.72 . ZL- 3.33
(7)
where ZL is the net charge of given nucleotide at
pH 7.5, and AGt is expressed in kJ/mol. Fig.5 illustrates that phosphates show similar trend ; their affinities increase when the ligand becomes more negatively
charged, but the dependence of AGE on ZL is more
complex.
Effect of Magnesium Ions on the Binding
of A T P to Aldolrrse
Fluorescence quenching of ansyl-aldolase complex by ATP was markedly changed upon addition
of Mg2' (Fig.6A). In control experiments it was
demonstrated that 1 mM MgCL or 2 mM NaCl
\
13
3.5
0
3.0
I
I
~
I
Id
AF/Fo
Fig. 6. Effect of magnesium ions on the binding of ATP to aldolase.
(A) Plot of relative fluorescence changes versus total Mg2+ concentration; Mg2+ concentrations were: (0) 0 mM,).( 0.32 mM,
and (A) 1.07 mM. (B) Data from A plotted against free Mg2+concentration
quenched only 3 % of the initial fluorescence of
enzyme-bound ansyl. In all experiments with Mg2+,
the concentration of chloride ions was kept at constant
level by addition of appriopriate amount of NaC1.
This concentration of chloride is well below the
inhibition constant for C1- [34]. Using Eqn (5) con-
449
A. A. Kasprzak and M. Kochman
2.5
1
0
DISCUSSION
4
8
12
16
20
[Fru-1.6-61 (FM)
24
28
Fig.7. Inhibition of aldolase by ATP. (A) Hanes-Woolf plot of the
data. (B) Dependence of K"mpp on the concentration of ATP
centrations of free ATP were calculated for each point
along the titration curve. Fig.6B shows that changes
in the fluorescence plotted against free ATP concentration follow almost the same curve, indicating that
the MgATP complex does not contribute to the displacement of ansyl molecules from the active site of
aldolase. Otherwise, fluorescence changes should differ
markedly when [Mg"] = 0 and 1 mM, i.e. under the
conditions where all added ATP is free or almost
totally complexed.
Binding of Fructose-1,6-Pz and ATP
to Glycerol-l-yhosphate Labelled o j AIdoluse
Ansyl, when bound to glycerol-2-phosphate derivative of aldolase displayed similar fluorescence
maximum as observed for the native enzyme (Fig.2).
The fluorescence intensity is 6.5-fold lower compared
to that for the native aldolase. On the addition of
fructose-l,6-Pz or ATP this fluorescence is decreased
by 20 - 25 %. The values of dissociation constants for
fructose-1,6-Pz and ATP obtained from fluorometric
titrations were 0.53 and 0.30 mM, respectively.
Kinetic Measurements
The effect of ATP on the kinetics of the fructose1,6-P2 cleavage reaction catalyzed by aldolase is
illustrated in Fig. 7. It was also demonstrated that the
inhibitory effect of 15 mM ATP could be entirely
reversed by 5 mM fructose-1,6-P2 whereas the enzymatic reaction was almost completely stopped by
15 mM ATP in the presence of a low substrate concentration (1.5 pM). It is apparent from Fig. 7 and the
data mentioned above that ATP is a pure competitive
inhibitor for the fructose-1,6-P2 cleavage reaction.
The value of K; obtained was 0.04 0.015 mM.
The results reported here represent the first direct
investigation on nucleotide binding sites on aldolase.
Our experiments have shown that each aldolase subunit has two nucleotide binding sites differing in their
affinity. Tight binding of nucleotides and phosphates
occurs at the active center since binding of ATP to
this site was eliminated in the presence of fructose1,6-P2 (Fig. 1C) and pure competitive inhibition was
observed with this nucleotide (Fig. 7). Furthermore,
the tight binding site could not be detected in the
glycerol-l-phosphate labelled aldolase in which catalytic site was covalently blocked (Fig. 1D).
Binding to the weak site is presumably associated
with a basic group with pK, below 8, since affinity of
this site for ATP was markedly reduced at pH 8.5
(Fig.1 B). From the available data it is difficult to
localize this site. The interaction of ATP with aldolase
in the presence of saturating concentration of fructose-1,6-P~might argue against the possibility that
the weak site is situated at the active center. However,
others reported that in the presence of fructose-l,6-P2
only 15 % of active sites were occupied by hexose bisphosphate and the remaining 85 % by dihydroxyacetone-P [35]. Thus the question whether the weak
site is the same one which binds C-6 phosphate of
the substrate remains still open.
Dissociation constants for ATP obtained by equilibrium dialysis, fluorescence quenching and kinetic
measurements are almost the same (Kd = 0.030.04 mM). These values are at least an order of
magnitude lower than that reported by Spolter et al.
[lo], who found a rather wide range of Ki for ATP
(0.5 - 1.4 mM). The discrepancy might arise from
different conditions under which their experiments
were performed. The above authors used a lower
pH (7.1) which entails the decrease of the negative
charge of terminal phosphate group of the nucleotide.
It is also not clear whether sulfate ions were removed
from their coupling enzymes mixture. Different pH
and the presence of inhibitory anions might significantly affect the affinity of the nucleotide to aldolase.
Among the nucleotides tested there are large
differences in their affinity to aldolase. It is evident
from Fig. 5 that factor underlying this selectivity in
the binding is the negative charge of the 5'-phosphate
group of the interacting ligand. The experiments with
magnesium ions are also consistent with this view.
The negative charge of the MgATP complex is two
units lower than for free nucleotide. This should decrease the affinity of MgATP approximately 100-fold.
On the other hand, for series of nucleotides, extrapolation of AGE to Z L = 0 predicts that non-electrostatic forces are also involved in this interaction.
Nevertheless, the dissection of AG; into the contributions of electrostatic, hydrophobic and other forces
450
A. A. Kasprzak and M. Kochman: Interaction of Aldolase with Mononucleotides
is hazardous. Previous studies on aldolase have shown
that standard free energy changes for similar ligands
can consist of quite different entropic and enthalpic
contributions [36]. Thus, free energy changes alone
might not provide much of an insight into the nature
of the interaction of adenine nucleotides with aldolase.
Results of experiments presented here have indicated that similarly to hexokinase [37], phosphofructokinase [38], phosphoglycerate kinase [39], and
pyruvate kinase [40], the subunit of aldolase contains
two sites to which adenine nucleotides can be bound.
It is also clear that although electrostatic forces are
essential for binding of nucleotides to aldolase, this
interaction cannot be considered as simple ionic
association in aqueous solution. We suppose that the
adenosine moiety, although contributing a little to
AGE,, may play a role in preventing an uncorrectly
positioned phosphate group of the nucleotide coming
into a direct contact with the positively charged
residue of the enzyme. Lack of interaction of CAMP
with aldolase and weak but detectable binding observed for adenosine support this hypothesis. It seems
reasonable to conclude that binding of nucleotides to
aldolase is a result of ‘thermodynamic selectivity’ and
‘structural specificity’ [41]. This led us to believe that
aldolase possesses a mononucleotide binding domain.
Additional evidence in support of this proposal is
provided by the observation that for homologous
rabbit liver aldolase, the adenosine moiety contributes
significantly to the binding of nucleotides (Kasprzak
and Kochman, unpublished results).
This work was supported by Polish Ministry of Science, Higher
Education and Technology, grant R 1.9.
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A. A. Kasprzak and M. Kochman, Instytut Chemii Organiczej i Fizycznej Politechniki Wroclawskiej,
Wybrzeie Wyspianskiego 27, PL-50-370 Wroclaw, Poland
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