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

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 dimet

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 K0.5 appfor K+and Na+were

190 and 64-fold lower than in water Ki app for K+and

Na+ decreased 116 and 135-fold between 20 and 40%

dimethylsulfoxide The ratios of Ki app/K0.5 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

Mg2+ exhibit high selectivity for their respective cation

binding sites, whereas when Na+ substitutes K+, Na+ and Mg2+bind with high affinity to their incorrect sites This is evident by the ratio of the affinities of Mg2+and

K+for the monovalent cation binding site, which is close

to 200 For Na+ and Mg2+ this ratio is approximately

20 Therefore, the data suggest that K+ induces con-formational changes that prevent the binding of Mg2+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 NH4+,

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 (CH3)4N+ 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 Mg2+-ATP, oxalate,

Mg2+, and either K+ 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)1more 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 NH4+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 Mg2+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 Mn2+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+, Mg2+

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: lramirez@laguna.fmedic.unam.mx

Abbreviations: V, limit velocity; K 0.5 , activator constant; K 0.5 app ,

apparent activator constant; h, Hill coeficient; k cat , catalytic constant;

K i app , 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

can bind to the site for K+and vice versa, in the absence of

Mg2+, K+can bind to the Mg2+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 lL of 50 mM Tris/HCl pH 7.6 The solutions were

passed twice through SephadexG-25 insulin centrifuge

columns [28] As noted elsewhere [29], the concentration of

contaminating NH4+, Na+or K+in the assay mixtures

was below the limits of detection (10 lM)

Assay of pyruvate kinase activity

The formation of pyruvate was measured at 25°C in a

coupled system with lactate dehydrogenase and NADH

[30] In water or in the binary water/dimethylsulfoxide

system, 1 mL of reaction mixture contained 1 mM

phos-phoenolpyruvate, 3 mM ADP, 3 mM MgCl2, 0.24 mM

NADH, 25 mM Tris/HCl pH 7.6 and the concentrations

of NaCl and KCl indicated in the Results and discussion

section To avoid ionic strength effects (CH3)4NCl was

added to give a final salt concentration of 100 mM In all

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)1pyruvate kinase, 25 mM Tris/HCl pH 7.6 and either K+, Na+or (CH3)4N+with or without 1 mMphosphoenolpyruvate and

3 mM Mg2+ 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)1Æcm)1 for pyruvate kinase [32] and 1.45 mLÆmg)1Æcm)1for lactate dehydrogenase [33]

Results and discussion

Comparative effect of Na+and K+on the activity

of pyruvate kinase The effect of Na+and K+concentration on the activity of pyruvate kinase was determined in mixtures with various dimethylsulfoxide concentrations; a saturating concentra-tion of Mg2+ was maintained constant (Fig 1) In the concentrations of Na+and K+that could be assayed in 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 Mg2+ 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

displacement of Mg2+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 K0.5 app

and Ki appfor both monovalent cations, it is relevant that

the decrease of the K0.5 appwas more important for K+than

for Na+; the ratio of K for K+in water to that in 40%

dimethylsulfoxide increased 190-fold, whereas with Na+the ratio increased 64-fold (Fig 2) Compared to the twofold to fourfold decrease in K0.5for 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 Ki app for Na+ and K+ at different cosolvent concentrations were compared At variance to the difference in K0.5 app for Na+and K+, it was found that dimethylsulfoxide affected to similar extents their respective

Ki app(Table 1) This was clearly evident in the ratios Ki app/

K0.5 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 Mg2+

To gain further insight into how dimethylsulfoxide affects the competition between the monovalent cations and Mg2+

in pyruvate kinase, we determined the kinetics of saturation for Na+and K+in presence of 1.35 and 6.46 mMMg2+free

(Fig 3) With 6.46 mMMg2+free, the Kappand V for Na+ were approximately four and two times higher than with 1.35 mMMg2+free; the Kappand V for K+were not affected

by Mg2+concentration It is noted that Mg2+did not affect the Ki appfor Na+, albeit, the Ki appfor K+was approxi-mately sixtimes higher at the higher Mg2+concentration The results indicate that in comparison to K+, Na+binds very tightly to the divalent cation binding site When Mg2+ 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 Mg2+binds more easily to the monovalent cation binding site in the

Na+-pyruvate kinase complexthan in the K+-pyruvate kinase complex

We carried out additional kinetic studies in order to further characterize the competition between Na+ and

Mg2+and that of K+ and Mg2+ In media with 40% dimethylsulfoxide, the kinetics of Na+ activation at

Mg2+freeconcentrations in the range of 1.35–9.38 mMwere determined For determination of the kinetics of Mg2+, the concentrations of Na+and K+were varied between 1.4 and 11.8 mMand from 6.46 to 33.14 mM, respectively The subsaturating regions of the curves were used to generate Lineweaver–Burk plots (Fig 4A–C) To calculate K and

Ki, the Kapp 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,

3 m M ADP, 3 m M MgCl 2 , 0.24 m 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% dimethyldimethyl-sulfoxide (r); 30% dimethyldimethyl-sulfoxide (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)1without monovalent cation

added, to 0.2 lgÆmL)1and 0.1 lgÆmL)1when 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)1Æmg 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 Mg2+ were

0.017 mM, 0.019 mM and 0.013 mM, respectively These

results indicate that in 40% dimethylsulfoxide the enzyme

binds with the same affinity Na+, K+and Mg2+ Under

conditions in which Na+ competes with Mg2+, the Ki

values for Na+and for Mg2+were 0.22 mMand 0.41 mM,

respectively In the competition of Mg2+with K+, the Ki

for Mg2+was 4.38 mM Therefore, in comparison to Na+

and Mg2+, there is a higher selectivity between K+ and

Mg2+for their respective sites This is clearly evident in the

ratios Ki Mg2+/KNa+, Ki Na+/KMg2+ and Ki K+/KMg2+

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 Mg2+ inhibition Another alternative is that in comparison to

K+, the similar ionic radii and bond distances of Na+and

Mg2+[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 0.5h+ 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

+ Sh+ (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 sixexperiments 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 i app 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)

k cat (s)1) K appa(m M ) h K i app (m M ) k cat (s)1) K appa(m M ) h K i app (m M )

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 activated by Na + (d) or K + (m) in water to

K 0.5 app at the indicated dimethylsulfoxide (DMSO) concentrations The ratios were cal-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.

respectively In this respect, the example of fructose

1,6-bisphosphatase is illustrative The small cation Li+,

displaces Mg2+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 (DDGs) is

predominantly governed by the enthalpy of solution

(DDH) [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°tof 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°tto the apparent binding energies of Na+and K+in pyruvate kinase For the latter calculations, we assumed that the

K0.5 app is equal to the Kd, 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°b¼ DG°b(water–dimethylsulfoxide)) DG°b(100% water)).For

K+, the DDG°b-values calculated in 20 and 40% dimethyl-sulfoxide were )6.86 and )13.02 kJÆmol)1; with Na+the

Fig 3 Effect of Mg2+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 , 1.35 m M (d) and 6.46 m 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°) 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 Mg2+ 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 Mg2+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 Mg2+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 + ) and Mg 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.

Mg2+free

(m M )

K app

(m M )

V (lmolÆmin)1Æmg)

Mg2+free

(m M )

K app

(m M )

V (lmolÆmin)1Æmg)

Na+ (m M )

K app

(m M )

V (lmolÆmin)1Æmg)

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+, Mg2+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 complexwith Mg2+and phosphoenolpyruvate in the presence of Na+, K+, or (CH3)4N+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 ) were taken and

transformed to kJÆmol)1from 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)

DG  t

(kJÆmol)1)

DG  b

a

(kJÆmol)1)

DG  t

(kJÆmol)1)

DG  b

a

(kJÆmol)1)

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)1in 25 m M Tris/HCl, pH 7.6, 1 m M

phosphoenolpyruvate, 3 m M Mg2+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) and 90 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) and 0.2 m M K + (s) were added In all cases, salt concentration (100 m M ) was kept constant by including appropriate amounts of (CH ) 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 (CH3)4N+, 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

(CH3)4N+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 Mg2+

exhibit high selectivity for their respective cation binding

sites, whereas Na+and Mg2+are promiscuous with high

affinity for their incorrect sites This is clearly evident in

the ratio of the affinities of Mg2+ and K+ for the

monovalent cation binding site, which is approximately

200 For Na+and Mg2+ 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 Mg2+

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°tand 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´o Patin˜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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