Báo cáo khoa học: Selectivity of pyruvate kinase for Na+ and K+ in water/dimethylsulfoxide mixtures docx

Selectivity of pyruvate kinase for Na
+
and K
+
in
water/dimethylsulfoxide mixtures
Leticia Ramı
´
rez-Silva and Jesu
´
s Oria-Herna
´
ndez
Departamento de Bioquı
´
mica, Facultad de Medicina, Universidad Nacional Auto
´
noma de Me
´
xico, Me
´
xico
In aqueous media, muscle pyruvate kinase is highly
selective for K
+
over Na
+
. We now studied the selec-
tivity of pyruvate kinase in water/dimethylsulfoxide mix-
tures by measuring the activation and inhibition constants
of K

+
and Na
+
, i.e. their binding to the monovalent and
divalent cation binding sites of pyruvate kinase, respect-
ively [Melchoir J.B. (1965) Biochemistry 4, 1518–1525]. In
40% dimethylsulfoxide the K
0.5 app
for K
+
and Na
+
were
190 and 64-fold lower than in water. K
iapp
for K
+
and
Na
+
decreased 116 and 135-fold between 20 and 40%
dimethylsulfoxide. The ratios of K
iapp
/K
0.5 app
for K
+
and Na
+
were 34–3.5 and 3.3–0.2, respectively. There-

fore, dimethylsulfoxide favored the partition of K
+
and
Na
+
into the monovalent and divalent cation binding
sites of the enzyme. The kinetics of the enzyme at sub-
saturating concentrations of activators show that K
+
and
Mg
2+
exhibit high selectivity for their respective cation
binding sites, whereas when Na
+
substitutes K
+
,Na
+
and Mg
2+
bind with high affinity to their incorrect sites.
This is evident by the ratio of the affinities of Mg
2+
and
K
+
for the monovalent cation binding site, which is close
to 200. For Na
+

and Mg
2+
this ratio is approximately
20. Therefore, the data suggest that K
+
induces con-
formational changes that prevent the binding of Mg
2+
to
the monovalent cation binding site. Circular dichroism
spectra of the enzyme and the magnitude of the transfer
and apparent binding energies of K
+
and Na
+
indicate
that structural arrangements of the enzyme induced by
dimethylsulfoxide determine the affinities of pyruvate
kinase for K
+
and Na
+
.
Keywords: dimethylsulfoxide; magnesium ion; potassium
ion; pyruvate kinase; sodium ion.
Rabbit muscle pyruvate kinase catalyzes the transfer of the
phosphoryl group of phosphoenolpyruvate to ADP. The
reaction is largely favored toward the formation of pyruvate
and ATP [1]. An important characteristic of pyruvate kinase
is that it has an absolute requirement for K

+
[2]. The
enzyme also catalyzes the reaction in the presence of NH
4
+
,
Rb
+
and Tl
+
, but the activity is 80–60% of that with K
+
;
with Na
+
and Li
+
, it is only 8% and 2%, respectively, and
with Tris [3] and (CH
3
)
4
N
+
it is 0.02% [4]. Although
monovalent cations support markedly different catalytic
activities, the attempts to correlate the catalytic activity with
changes in structural properties such as immunoelectro-
phoretic patterns [5], ultraviolet absorption [6], and circular
dichroism of the enzyme [7] have failed. In contrast to

dialkylglycine decarboxylase, where the substitution of K
+
for Na
+
causes distinct structural changes [8], the structure
of pyruvate kinase cocrystallized with Mg
2+
-ATP, oxalate,
Mg
2+
,andeitherK
+
or Na
+
exhibits only subtle
differences [9]. Likewise, the similarities in the coordination
number, the polarizability, and stereochemistry of the Na
+
and K
+
ligand interactions do not account for the marked
discrimination of pyruvate kinase for these cations [10–13].
An important contributing factor in the selectivity for
Na
+
and K
+
may be the difference in the dehydration free
energy required for stripping the water molecules from the
two cations. It is 85 kJÆmol

)1
more favorable for K
+
[10,14–
16]. In fact, when pyruvate kinase was entrapped in reverse
micelles with low water content, it was found that Na
+
was
an effective activator of the enzyme [17], and that its
effectiveness was comparable to that of NH
4
+
and Rb
+
.
This suggested that partition of Na
+
into the activating site
is hindered by energetic barriers [18], and that a low water
environment favored the transfer of Na
+
into pyruvate
kinase. Nevertheless, under all conditions, it was observed
that at equivalent concentrations of K
+
and Na
+
,the
activity of pyruvate kinase was always higher with K
+

[17].
Pyruvate kinase also requires two Mg
2+
ions per active
site for activity [19]. One binds directly to the protein in the
absence of substrates (site I) [20] and the other to the
nucleotide substrate site (site II) [19]. Other divalent cations
may substitute them; ions smaller than Mn
2+
bind prefer-
entially to site II, whilst the larger ions bind to site I [21].
However, regarding the characteristics of the sites for
divalent and monovalent cations it has been shown that the
sites are not entirely specific. In the absence of K
+
,Mg
2+
Correspondence to L. Ramı
´
rez-Silva, Departamento de Bioquı
´
mica,
Facultad de Medicina, Apartado Postal 70–159, Universidad
Nacional Auto
´
noma de Me
´
xico, 05410 Me
´
xico D.F., Me

´
xico.
Fax: + 525 6162419, Tel.: + 525 6232510,
E-mail:
Abbreviations: V,limitvelocity;K
0.5
, activator constant; K
0.5 app
,
apparent activator constant; h,Hillcoeficient;k
cat
, catalytic constant;
K
iapp
, apparent inhibition constant; K
i
, inhibition constant; K
app
,
apparent Michaelis–Menten constant; K, Michaelis–Menten constant.
Enzymes: lactate dehydrogenase (EC 1.1.1.27); pyruvate kinase
(EC 2.7.1.40).
(Received 18 February 2003, revised 31 March 2003,
accepted 4 April 2003)
Eur. J. Biochem. 270, 2377–2385 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03605.x
canbindtothesiteforK
+
and vice versa, in the absence of
Mg
2+

,K
+
canbindtotheMg
2+
binding site [22]. This is of
importance to the kinetics of the enzyme, as the occupancy
of the two sites by either monovalent or divalent cations
yields an inactive enzyme, or an enzyme with very low
activity [23].
To ascertain the factors that control the selectivity of the
two-cation binding sites, the kinetic and structural differ-
ences of pyruvate kinase with K
+
and Na
+
were studied. A
limitation in the study of the activation of pyruvate kinase
by Na
+
is the low affinity that the enzyme exhibits for this
monovalent cation [17]. Reports on the Na
+
/K-ATPase
[24–27] show that the apparent affinities of the enzyme for
both Na
+
and K
+
are higher in water/dimethylsulfoxide
mixtures. The authors indicate that dimethylsulfoxide

favors a conformation of the enzyme that exhibits higher
apparent affinities for the monovalent cations. Therefore,
it was thought that water/dimethylsulfoxide mixtures could
be used to determine if energetic barriers or structural
arrangements of the enzyme control the selectivity of
pyruvate kinase for Na
+
and K
+
. This work shows that
dimethylsulfoxide induced structural arrangements that
favored the partition of both cations into the enzyme. It
was also found that in water/dimethylsulfoxide mixtures,
K
+
binds preferentially to the monovalent cation binding
site, whereas the smaller cation Na
+
binds to the divalent
cation (inhibitory) binding site with a higher affinity
(20-fold) than K
+
.
Materials and methods
Materials
Rabbit muscle pyruvate kinase and hog muscle lactate
dehydrogenase were obtained as ammonium sulfate sus-
pensions from Boehringer. The cyclohexylammonium salts
of ADP and phosphoenolpyruvate were from Sigma.
NADH sodium salt was converted to the cyclohexyl-

ammonium salt by ion exchange following the protocol of
the manufacturer (Sigma). Analytical and spectroscopy
grades of dimethylsulfoxide were from Merck. Prior to the
experiments, the suspensions of pyruvate kinase and lactate
dehydrogenase were centrifuged and the pellets dissolved
in 90 lLof50m
M
Tris/HCl pH 7.6. The solutions were
passed twice through Sephadex G-25 insulin centrifuge
columns [28]. As noted elsewhere [29], the concentration of
contaminating NH
4
+
,Na
+
or K
+
in the assay mixtures
was below the limits of detection (10 l
M
).
Assay of pyruvate kinase activity
The formation of pyruvate was measured at 25 °Cina
coupled system with lactate dehydrogenase and NADH
[30]. In water or in the binary water/dimethylsulfoxide
system, 1 mL of reaction mixture contained 1 m
M
phos-
phoenolpyruvate, 3 m
M

ADP, 3 m
M
MgCl
2
,0.24m
M
NADH, 25 m
M
Tris/HCl pH 7.6 and the concentrations
of NaCl and KCl indicated in the Results and discussion
section. To avoid ionic strength effects (CH
3
)
4
NCl was
added to give a final salt concentration of 100 m
M
.Inall
experiments, the overall mixture contained 10, 15, 20, 40, 45,
50 and 60 lg of lactate dehydrogenase when mixtures 0, 5,
10, 20, 25, 30 and 40% dimethylsulfoxide (w/v), were,
respectively, used; this was carried out because the activity
of lactate dehydrogenase is inhibited by dimethylsulfoxide.
The specific activity of pyruvate kinase in water/dimethyl-
sulfoxide mixtures was not increased by the fivefold
inclusion of lactate dehydrogenase. Activity was initiated
by introducing pyruvate kinase.
Fluorescence experiments
Fluorescence emission spectra of pyruvate kinase in 100%
water and in various water/dimethylsulfoxide mixtures were

determined in an ISS PCI Photon Counting Spectro-
fluorometer (ISS, Urbana, Il) thermoregulated at 25 °C.
Excitation wavelength was set at 295 nm with excitation
and emission slits of 4 nm. Emission was measured from
300 to 450 nm. The samples contained 30 lgÆmL
)1
pyruvate
kinase, 25 m
M
Tris/HCl pH 7.6 and either K
+
,Na
+
or
(CH
3
)
4
N
+
with or without 1 m
M
phosphoenolpyruvate and
3m
M
Mg
2+
. The fluorescence spectra of blanks (no
protein) were subtracted from those that contained the
enzyme. From the difference, the spectral center of mass, or

average emission wavelength was calculated with the
software provided by ISS Inc. as indicated elsewhere [31].
Circular dichroism experiments
CD measurements were carried out on a Jasco J-720
spectropolarimeter. A 5-mm quartz cell was used for near-
ultraviolet (UV) CD experiments. The experiments were
conducted at room temperature. The concentration of
pyruvate kinase was 1 mgÆmL
)1
. Spectral scans were run
from 270 to 300 nm at intervals of 0.5 nm and a time
constant of 5 s. The spectra of blanks were subtracted from
those that contained the protein. CD is expressed as molar
ellipticity.
Protein concentrations were determined by measuring
the absorbance at 280 nm using the absorption coefficients
of 0.54 mLÆmg
)1
Æcm
)1
for pyruvate kinase [32] and
1.45 mLÆmg
)1
Æcm
)1
for lactate dehydrogenase [33].
Results and discussion
Comparative effect of Na
+
and K

+
on the activity
of pyruvate kinase
TheeffectofNa
+
and K
+
concentration on the activity of
pyruvate kinase was determined in mixtures with various
dimethylsulfoxide concentrations; a saturating concentra-
tion of Mg
2+
was maintained constant (Fig. 1). In the
concentrations of Na
+
and K
+
thatcouldbeassayedin
100% water, K
+
induced a strong activation that exhibited
saturation kinetics; Na
+
induced a relatively small enhance-
ment of activity that was far from saturation. In water/
dimethylsulfoxide mixtures the titration curves were bipha-
sic; the progressive increase in activity was followed by
inhibition. The increase in activity is due to the progressive
occupancy of the monovalent cation binding site by Na
+

or
K
+
to an enzyme that has the site for divalent cation filled
with Mg
2+
. According to Buchbinder and Reed [21],
Na
+
and K
+
bind to the divalent cation binding site I.
Thus, the inhibition shown in Fig. 1 would result from the
2378 L. Ramı
´
rez-Silva and J. Oria-Herna
´
ndez (Eur. J. Biochem. 270) Ó FEBS 2003
displacement of Mg
2+
by relatively high concentrations of
the monovalent cations [3,17,22,23].
A notable feature of the data in Fig. 1 is that as
dimethylsulfoxide concentration was increased, the curves
shifted to the left. This is because the concentration of Na
+
and K
+
required for half-maximal activation and inhibition
decreased. The kinetic constants at the various dimethyl-

sulfoxide concentrations are in Table 1. Although increas-
ing dimethylsulfoxide concentrations decreased the K
0.5 app
and K
iapp
for both monovalent cations, it is relevant that
the decrease of the K
0.5 app
was more important for K
+
than
for Na
+
;theratioofK
0.5 app
for K
+
inwatertothatin40%
dimethylsulfoxide increased 190-fold, whereas with Na
+
the
ratio increased 64-fold (Fig. 2). Compared to the twofold to
fourfold decrease in K
0.5
for Na
+
and K
+
in the Na
+

/K-
ATPase [24–27], this increase in affinities of pyruvate kinase
for monovalent cations is the highest reported in the
presence of dimethylsulfoxide and is similar to that previ-
ously found in reverse micelles [17].
In order to explore how dimethylsulfoxide affects the
partition of Na
+
and K
+
into the divalent metal cation
binding site, the K
iapp
for Na
+
and K
+
at different
cosolvent concentrations were compared. At variance to the
difference in K
0.5 app
for Na
+
and K
+
,itwasfoundthat
dimethylsulfoxide affected to similar extents their respective
K
iapp
(Table 1). This was clearly evident in the ratios K

iapp
/
K
0.5 app
(Fig. 2, inset). For Na
+
, this ratio varied from 3.3 in
20% dimethylsulfoxide to 0.2 in 40% dimethylsulfoxide,
whereas with K
+
, this decreased from 34 to 3.5 in the same
range of dimethylsulfoxide concentrations. The decrease in
the ratio indicates that the partition of Na
+
and K
+
into
the divalent cation binding site is more sensitive to
dimethylsulfoxide than the partition of Na
+
and K
+
into
the monovalent cation binding site. However in 20–40%
dimethylsulfoxide, K
+
binds preferentially to the mono-
valent cation binding site, whereas Na
+
binds with similar

affinity to both, monovalent and divalent cation binding
sites (Fig. 2, inset).
Competition between Na
+
or K
+
and Mg
2+
To gain further insight into how dimethylsulfoxide affects
the competition between the monovalent cations and Mg
2+
in pyruvate kinase, we determined the kinetics of saturation
for Na
+
and K
+
in presence of 1.35 and 6.46 m
M
Mg
2+
free
(Fig. 3). With 6.46 m
M
Mg
2+
free
,theK
app
and V for Na
+

were approximately four and two times higher than with
1.35 m
M
Mg
2+
free
;theK
app
and V for K
+
were not affected
by Mg
2+
concentration. It is noted that Mg
2+
did not affect
the K
iapp
for Na
+
, albeit, the K
iapp
for K
+
was approxi-
mately six times higher at the higher Mg
2+
concentration.
The results indicate that in comparison to K
+

,Na
+
binds
very tightly to the divalent cation binding site. When Mg
2+
replaces Na
+
from the divalent cation binding site, the
inhibitory effect of Na
+
is released and the maximal activity
increased by twofold. It is also relevant that Mg
2+
binds
more easily to the monovalent cation binding site in the
Na
+
-pyruvate kinase complex than in the K
+
-pyruvate
kinase complex.
We carried out additional kinetic studies in order to
further characterize the competition between Na
+
and
Mg
2+
andthatofK
+
and Mg

2+
.Inmediawith40%
dimethylsulfoxide, the kinetics of Na
+
activation at
Mg
2+
free
concentrations in the range of 1.35–9.38 m
M
were
determined. For determination of the kinetics of Mg
2+
,the
concentrations of Na
+
and K
+
were varied between 1.4
and 11.8 m
M
and from 6.46 to 33.14 m
M
, respectively. The
subsaturating regions of the curves were used to generate
Lineweaver–Burk plots (Fig. 4A–C). To calculate K and
K
i
,theK
app

values derived from the data in Fig. 4A–C
were replotted against the concentrations of the inhibitor
(Fig. 4D–F). In all cases, the double reciprocal plots showed
a competitive pattern. The linear fit of the replots indicates
that inhibition is purely competitive.
Fig. 1. Effect of Na
+
(A) and K
+
(B) on the activity of pyruvate kinase
in 100% water and in various water/dimethylsulfoxide mixtures. One
millilitre of reaction mixture contained 1 m
M
phosphoenolpyruvate,
3m
M
ADP, 3 m
M
MgCl
2
,0.24m
M
NADH, 25 m
M
Tris/HCl pH 7.6
and 10–60 lg lactate dehydrogenase. The concentrations of Na
+
and
K
+

were varied as indicated and (CH
3
)
4
NCl was added in each case to
give a final salt concentration of 100 m
M
in order to maintain constant
ionic strength. The activities in the presence of the indicated concen-
trations of the logarithm of NaCl and KCl in 100% water (d); 5%
dimethylsulfoxide (h); 10% dimethylsulfoxide (.); 20% dimethyl-
sulfoxide (n); 25% dimethylsulfoxide (r); 30% dimethylsulfoxide (s)
and 40% dimethylsulfoxide (j) are shown. The reaction was started
by the addition of pyruvate kinase. Different amounts of pyruvate
kinase were used ranging from 70 lgÆmL
)1
without monovalent cation
added, to 0.2 lgÆmL
)1
and 0.1 lgÆmL
)1
when the mixtures contained
Na
+
and K
+
, respectively. The basal cation-independent activities
(0.025, 0.041, 0.15, 1.1, 3, 5.9 and 19.3 lmolÆmin
)1
Æmg for 0%, 5%,

10%, 20%, 25%, 30% and 40% (w/v) dimethylsulfoxide, respectively)
were subtracted. The temperature was 25 °C. The mean of three to six
experiments is shown. The standard deviations are shown in Table 1.
Ó FEBS 2003 Selectivity of pyruvate kinase for Na
+
and K
+
(Eur. J. Biochem. 270) 2379
The kinetic constants resulting from Fig. 4 are shown
in Table 2. The K values for Na
+
,K
+
and Mg
2+
were
0.017 m
M
, 0.019 m
M
and 0.013 m
M
, respectively. These
results indicate that in 40% dimethylsulfoxide the enzyme
binds with the same affinity Na
+
,K
+
and Mg
2+

. Under
conditions in which Na
+
competes with Mg
2+
,theK
i
values for Na
+
and for Mg
2+
were 0.22 m
M
and 0.41 m
M
,
respectively. In the competition of Mg
2+
with K
+
,theK
i
for Mg
2+
was 4.38 m
M
. Therefore, in comparison to Na
+
and Mg
2+

, there is a higher selectivity between K
+
and
Mg
2+
for their respective sites. This is clearly evident in the
ratios K
i Mg2+
/K
Na+
, K
iNa+
/K
Mg2+
and K
iK+
/K
Mg2+
that are 24, 17 and 231, respectively.
The overall results must be explained in terms of the
intrinsic differences between Na
+
and K
+
. Because the V
with K
+
is always higher than with Na
+
in all experimental

conditions (Tables 1 and 2), it is possible that K
+
gives rise
to a particular conformation in order to prevent Mg
2+
inhibition. Another alternative is that in comparison to
K
+
, the similar ionic radii and bond distances of Na
+
and
Mg
2+
[11,16], account for their ability to occupy with high
affinity the divalent and monovalent cation binding sites,
Table 1. Kinetics of the activation of pyruvate kinase by Na
+
and K
+
in 100% water and in various water/dimethylsulfoxide mixtures. The program
MICROCAL
-
ORIGIN
version 3.73 was used to calculate the apparent kinetic constants from the data of Fig. 1. The sigmoidal data were fitted to
v ¼ VÆS
h
/K
0.5
h
+ S

h
. Data that showed inhibition were fitted to v ¼ VÆS/K + S +(S)
2
/K
i
and those that exhibited both sigmoidicity and
inhibition were fitted to v ¼ VÆS
h
/K
0.5
h
+ S
h
+(S)
2
/K
i
. The fit of various curves to the Hill model was better than when adjusted to the Michaelis–
Menten equation, which may be due to the decrease in affinities for phosphoenolpyruvate and ADP-Mg
2+
when K
+
is nearly or totally absent [30].
The mean and standard deviations from three to six experiments are shown. The kinetic constants estimated for Na
+
in 100% water and 40%
dimethylsulfoxide exhibit large standard deviations. This is because in 100% water, the concentration of Na
+
required for half-maximal activation
was higher than the value that could be experimentally assayed; thus the values shown derive from large extrapolation of the experimental data. In

40% dimethylsulfoxide, the K
iapp
for Na
+
is approximately sixfold smaller than the K
app
for Na
+
, making it difficult to obtain a good fit of the
experimental points.
Dimethylsulfoxide
(%, w/v)
Na
+
K
+
k
cat
(s
)1
) K
app
a
(m
M
) hK
i app
(m
M
) k

cat
(s
)1
) K
app
a
(m
M
) hK
i app
(m
M
)
0 362 ± 150 179 ± 86 1.39 ± 0.14 – 1592 ± 296 21 ± 6 1.22 ± 0.10 142 ± 69
5 339 ± 23 58 ± 6 1.44 ± 0.09 – 2287 ± 90 23 ± 3 – 88 ± 14
10 427 ± 25 46 ± 4 1.46 ± 0.09 – 1714 ± 75 8 ± 0.7 – 92 ± 11
20 604 ± 43 20 ± 2 1.53 ± 0.02 65 ± 12 972 ± 19 1.3 ± 0.07 – 44 ± 2.4
25 695 ± 43 12 ± 1 1.19 ± 0.07 33 ± 9 1019 ± 23 0.76 ± 0.06 – 15.9 ± 1.2
30 604 ± 43 7 ± 0.6 – 16.8 ± 1.9 1157 ± 28 0.33 ± 0.02 – 4.5 ± 0.3
40 569 ± 316 2.8 ± 1.8 – 0.48 ± 0.32 466 ± 59 0.11 ± 0.03 – 0.38 ± 0.08
a
K
app
represents the K
0.5 app
and K
app
for the data fitted to the Hill and to the Michaelis–Menten equations, respectively.
Fig. 2. Ratios of K
0.5 app

for pyruvate kinase
activatedbyNa
+
(d)orK
+
(m)inwaterto
K
0.5 app
at the indicated dimethylsulfoxide
(DMSO) concentrations. Theratioswerecal-
culated from the data in Table 1. The inset
illustrates the ratios of the apparent inhibition
constants to K
0.5 app
at the indicated water/
dimethylsulfoxide mixtures. The data point
indicated by (j) represents the normalized
K
0.5 app
of pyruvate kinase with Na
+
or K
+
in
100% water.
2380 L. Ramı
´
rez-Silva and J. Oria-Herna
´
ndez (Eur. J. Biochem. 270) Ó FEBS 2003

respectively. In this respect, the example of fructose
1,6-bisphosphatase is illustrative. The small cation Li
+
,
displaces Mg
2+
from its divalent cation binding site, but it is
unable to occupy the K
+
binding site [34–36].
Solvation and energetic of binding of Na
+
and K
+
to pyruvate kinase
It has been hypothesized [18] that the difference between
Na
+
and K
+
in the activation and inhibition of pyruvate
kinase could be related to their different solvation energy.
Accordingly, we examined if the energy of transfer of
Na
+
and K
+
between solvents correlates with their
ability to affect the kinetics of pyruvate kinase. Due to
solvent–solvent interactions, water/dimethylsulfoxide mix-

tures are more structured than water or dimethylsulfoxide
alone [37–40]. In such mixtures, small cations like Na
+
show positive enthalpies of transfer from water to
mixtures with low dimethylsulfoxide concentrations.
Maximum desolvation of Na
+
occurs with approximately
30% dimethylsulfoxide (v/v); at higher dimethylsulfoxide
concentrations, Na
+
becomes more strongly solvated
due to strong ion–dipole interactions [41]. These changes
in ion solvation reflect on the free energy of transfer
between solvents. Indeed, when ion transfer is between
organic solvents, the free energy of solution (DDG
s
)is
predominantly governed by the enthalpy of solution
(DDH
s
) [41]. However, when water is one of the solvents,
entropy changes (TDS) contribute strongly to the free
energy of transfer [41].
In the light of the latter data, we explored whether there is
a relationship between the transfer energies of Na
+
and K
+
from water to water/dimethylsulfoxide and the affinity for

Na
+
and K
+
of pyruvate kinase. As shown in Table 3, the
transfer energies of monovalent cations become increasingly
negative as dimethylsulfoxide concentration is increased;
this reflects the solvation of cations in such media [42].
Table 3 also shows that the DG°
t
of both, Na
+
and K
+
from
water to dimethylsulfoxide mixtures are negative, which
indicates that the monovalent cations are more solvated in
water/dimethylsulfoxide mixtures than in 100% water.
However, the data also show that the DG°
t
is far more
negative with Na
+
than with K
+
. The latter data suggested
that the lower solvation of K
+
would favor its partition into
pyruvate kinase. Thus, we compared the values of DG°

t
to
the apparent binding energies of Na
+
and K
+
in pyruvate
kinase. For the latter calculations, we assumed that the
K
0.5 app
is equal to the K
d
, and from the equilibrium
constants we calculated the apparent binding energies.
The effect of dimethylsulfoxide on the affinity of pyruvate
kinase for both cations can be calculated from the difference
of apparent binding energies in water/dimethylsulfoxide
mixtures minus the values obtained in 100% water
(DDG°
b
¼ DG°
b
(water–dimethylsulfoxide)
) DG°
b
(100% water)
).For
K
+
,theDDG°

b
-values calculated in 20 and 40% dimethyl-
sulfoxide were )6.86 and )13.02 kJÆmol
)1
;withNa
+
the
Fig. 3. Effect of Mg
2+
on the activity of pyruvate kinase at various concentrations of Na
+
(A) and K
+
(B) in 40% dimethylsulfoxide. The
experimental conditions were as in Fig 1, except that the experiments were performed in the presence of 40% dimethylsulfoxide and two
concentrations of Mg
2+
free
,1.35m
M
(d)and6.46m
M
(s). The basal cation-independent activities were subtracted. The temperature was 25 °C.
The kinetic constants were calculated as in Table 1. The average from two experiments is shown.
Ó FEBS 2003 Selectivity of pyruvate kinase for Na
+
and K
+
(Eur. J. Biochem. 270) 2381
values were less negative )5.40 and )10.29 kJÆmol

)1
.As
shown in Table 3, the transfer energies are much less
negative than the apparent binding energies (DG°
b
)
.
This
indicates that solvation energy is not the predominant factor
in the control of the affinity for Na
+
and K
+
in pyruvate
kinase.
Fig. 4. Competition between Na
+
or K
+
and Mg
2+
. One millilitre of reaction mixture contained 1 m
M
phosphoenolpyruvate, 3 m
M
ADP, 0.24 m
M
NADH and 25 m
M
Tris/HCl pH 7.6. The temperature was 25 °C. The free concentrations of Mg

2+
were calculated using the Mg
2+
-ADP
association constants reported for aqueous media with the computer program
CHELATOR
[45]. Na
+
varied from 0 to 10 m
M
at various Mg
2+
free
fixed concentrations: 1.35 m
M
(m), 3.7 m
M
(h), 6.46 m
M
(d) and 9.38 m
M
(,). K
+
varied from 0 to 10 m
M
at various fixed Mg
2+
concentrations:
6.46 m
M

(m), 15.24 m
M
(h), 24.17 m
M
(d), and 33.14 m
M
(,). Conversely, Mg
2+
free
varied from 0.014 to 12.3 m
M
at various fixed Na
+
concentrations: 1.4 m
M
(m), 2.8 m
M
(h), 5.75 m
M
(d) and 11.2 m
M
(,). The subsaturating regions of the curves were used to generate the
Lineweaver–Burk plots (A–C). The K
app
values derived from these figures were plotted vs. the concentrations of the inhibitor (D–F). The kinetic
constants are shown in Table 3.
Table 2. Kinetic constants derived from competitive inhibition plots between monovalent cations (Na
+
and K
+

)andMg
2+
in 40% (w/v) dimethyl-
sulfoxide. The apparent kinetic constants were calculated from data of Fig. 4, A–C, except that the activity without monovalent cation was
not subtracted. The average from two experiments is shown. The standard error was approximately 5% in all cases. The K and K
i
were calculated
from the replots fitted to K
app
¼ K/K
i
[I] + K (Fig. 4, D–F). The linear correlation coefficients were above 0.995.
Na
+
K
+
Mg
2+
Mg
2+
free
(m
M
)
K
app
(m
M
)
V

(lmolÆmin
)1
Æmg)
Mg
2+
free
(m
M
)
K
app
(m
M
)
V
(lmolÆmin
)1
Æmg)
Na
+
(m
M
)
K
app
(m
M
)
V
(lmolÆmin

)1
Æmg)
1.35 0.086 45 6.46 0.048 73 1.4 0.11 38
3.70 0.158 44 15.24 0.085 73 2.8 0.15 39
6.46 0.273 45 24.17 0.115 66 5.75 0.36 34
9.38 0.416 41 33.14 0.165 78 11.8 0.69 39
K (m
M
) 0.017 K (m
M
) 0.019 K (m
M
) 0.013
K
i
(m
M
) 0.41 K
i
(m
M
) 4.38 K
i
(m
M
) 0.22
2382 L. Ramı
´
rez-Silva and J. Oria-Herna
´

ndez (Eur. J. Biochem. 270) Ó FEBS 2003
Conformation of pyruvate kinase in water and 40%
dimethylsulfoxide solutions
In the light of the latter data it was considered that the
different catalytic activities of pyruvate kinase with Na
+
and K
+
could be related to distinct structural features of the
enzyme. Therefore, we determined the intrinsic fluorescence
spectra of pyruvate kinase with and without cations and
various concentrations of dimethylsulfoxide. Differences in
the intrinsic fluorescence of pyruvate kinase incubated with
and without ligands (K
+
,Mg
2+
and phosphoenolpyruvate)
have been reported previously, and it has been proposed
that the distinct spectra correspond to the active and
inactive conformations of pyruvate kinase [17,29,43]. The
spectral center of mass of pyruvate kinase and pyruvate
kinase in complex with Mg
2+
and phosphoenolpyruvate in
the presence of Na
+
,K
+
,or(CH

3
)
4
N
+
were recorded in
mixtures that contained 0%, 5%, 10%, 20%, 30% and 40%
(w/v) dimethylsulfoxide. In agreement with previous data
[31], the recordings showed that in presence of 30 and 40%
dimethylsulfoxide and in the absence of ligands, pyruvate
kinase exhibited the active conformation. Moreover, in the
presence of dimethylsulfoxide and ligands, pyruvate kinase
continued to exhibit the active conformation; this was
Table 3. Transfer energies for K
+
and Na
+
from 100% water to water/
dimethylsulfoxide solutions, and apparent binding energies for pyruvate
kinase-K
+
and pyruvate kinase-Na
+
complexes in water and water/
dimethylsulfoxide mixtures. Transfer energies (DG°
t
)weretakenand
transformedtokJÆmol
)1
from the data of Kundu and Das (1979) [42].

The apparent binding energies (DG°
b
) were calculated from the data of
Table 1. Transfer energies were calculated in water/dimethylsulfoxide
mixtures by volume (v/v), and the apparent binding energies in water/
dimethylsulfoxide by weight (w/v).
Dimethylsulfoxide
(%, w/v)
Na
+
K
+
DG

t
(kJÆmol
)1
)
DG

b
a
(kJÆmol
)1
)
DG

t
(kJÆmol
)1

)
DG

b
a
(kJÆmol
)1
)
0–)4.27 – )9.58
20 )1.72 )9.67 )1.30 )16.44
40 )3.00 )14.56 )2.01 )22.6
a
Apparent binding energies were calculated assuming that the
K
0.5 app
is equal to K
d
.
Fig. 5. Near-UV-CD spectra of pyruvate kinase in aqueous media (A) and 40% dimethylsulfoxide (w/v) (B) in the presence of (CH
3
)
4
N
+
,Na
+
and
K
+
. The spectra were obtained in mixtures that contained pyruvate kinase at a concentration of 1 mgÆmL

)1
in 25 m
M
Tris/HCl, pH 7.6, 1 m
M
phosphoenolpyruvate, 3 m
M
Mg
2+
and the monovalent cation indicated. Na
+
and K
+
were added at concentrations in which maximal activation
of the enzyme was achieved. (A) 100 m
M
(CH
3
)
4
N
+
(h), 100 m
M
Na
+
(n)and90 m
M
K
+

(s) were included in the reaction mixtures, (B) 100 m
M
(CH
3
)
4
N
+
(h), 1.5 m
M
Na
+
(n)and0.2m
M
K
+
(s) were added. In all cases, salt concentration (100 m
M
) was kept constant by including
appropriate amounts of (CH
3
)
4
N
+
.
Ó FEBS 2003 Selectivity of pyruvate kinase for Na
+
and K
+

(Eur. J. Biochem. 270) 2383
independent of which monovalent cation was in the media.
It is also relevant that in the latter condition, dimethylsulf-
oxide did not modify the spectra (data not shown).
Previous attempts to find if there is a correlation between
the activating effect of various monovalent cations and
structural alterations of the enzyme have been unsuccessful
[7]. These studies have been performed in water and in
presence of K
+
,Li
+
and (CH
3
)
4
N
+
, but not Na
+
. Here,
we examined the effect of Na
+
and K
+
on the circular
dichroism spectra of pyruvate kinase in water media and in
40% dimethylsulfoxide. The concentrations of Na
+
and

K
+
that induced maximal activation were used (Fig. 5). In
all cases, the enzyme showed transition bands at 282 and
289 nm where Tyr and Trp residues overlap [44]. The
intensities of the spectra with the nonactivating cation
(CH
3
)
4
N
+
were very similar in water and 40% dimethyl-
sulfoxide. In the presence of K
+
and Na
+
, however, the
intensities of the spectra were, respectively, 25% and 17%
lower in 40% dimethylsulfoxide than in water. It is also
noteworthy, that in the presence of any cation, the CD
bands at 282 nm and 289 nm were sharper in 40%
dimethylsulfoxide than in aqueous media. This suggests
that in 100% water, the CD spectra reflect the average of
different conformations, whereas in dimethylsulfoxide there
would seem to be an enrichment of enzymes with the same
conformation.
Conclusions
The results of this study explain the high discrimination
of pyruvate kinase for Na

+
and K
+
.K
+
and Mg
2+
exhibit high selectivity for their respective cation binding
sites, whereas Na
+
and Mg
2+
are promiscuous with high
affinity for their incorrect sites. This is clearly evident in
the ratio of the affinities of Mg
2+
and K
+
for the
monovalent cation binding site, which is approximately
200. For Na
+
and Mg
2+
this ratio is approximately 20.
In the light of kinetic data, Na
+
and K
+
bind to the

monovalent binding site with the same affinity. However
the catalytic rates with K
+
are always higher; this
suggests that K
+
induces particular conformational
changes that are favorable for catalysis and that at the
same time prevent inhibition by Mg
2+
.
In regard to ionic selectivity, the physical characteristics
of the ions (ionic radius, coordination number, geometry)
and their solvation energy must be taken into account.
The differences in ionic radii of K
+
and Na
+
undoubtedly contribute to the selectivity of monovalent
and divalent cation binding sites in pyruvate kinase. As
to the energetics of binding, we have explored if the
magnitude of the transfer energies of Na
+
and K
+
from
water to dimethylsulfoxide is related to the apparent
binding energies of Na
+
and K

+
to pyruvate kinase (in
40% dimethylsulfoxide the DG°
t
and DG°
b
are )3.0 and
)14.56 kJÆmol
)1
for Na
+
and )2.01 and )22.6 kJÆmol
)1
for K
+
). We found that in presence of dimethylsulfoxide,
the factor that determines the affinity and selectivity of
pyruvate kinase for K
+
and Na
+
is not dehydration.
Instead, intrinsic fluorescence and near ultraviolet CD
studies show that dimethylsulfoxide induces structural
arrangements in which the whole enzyme population
acquires the high affinity active conformation.
Acknowledgements
The authors thank Rocı
´
oPatin

˜
o from Instituto de Quı
´
mica, Univer-
sidad Nacional Auto
´
noma de Me
´
xico for technical assistance in the CD
measurements and A. Go
´
mez-Puyou and M. Tuena de Go
´
mez-Puyou
for valuable suggestions, discussion and revision of the manuscript.
This work was partially supported by grants IN227202-3 from
DGAPA-UNAM and 32033-N from Consejo Nacional de Ciencia y
Tecnologı
´
a, Me
´
xico.
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(Eur. J. Biochem. 270) 2385