Eur. J. Biochem. 164,661 -666 (1987) 0 FEBS 1987

Changes in the quaternary structure of phosphoenolpyruvate carboxylase induced by ionic strength affect its catalytic activity Richard WAGNER, Daniel H. GONZALEZ, Florencio E. PODESTA and Carlos S. ANDRE0 Centro de Estudios Fotosintkticos y Bioquimicos, Rosario

(Received November 18/December 22, 1986) - EJB 86 1220

Phosphoenolpyruvate carboxylase from maize leaves dissociated into dimers and/or monomers when exposed to increasing ionic strength (e. g. 200 - 400 mM NaC1) as indicated by gel filtration experiments. Changes in the oligomerization state were dependent on pH, time of preincubation with salt and protein concentration. A dissociation into dimers and monomers was observed at pH 8, while at pH 7 dissociation into the dimeric form only was observed. Exposure of the enzyme to higher ionic strength decreased the activity in a time-dependent manner. Turnover conditions and glucose 6-phosphate protected the carboxylase from the decay in activity, which was faster at pH 7 than at pH 8. The results suggest that changes in activity of the enzyme, following exposure to high ionic strength, are the consequence of dissociation. Tetrameric and dimeric forms of the phosphoenolpyruvate carboxylase seemingly reveal different catalytic properties. We suggest that the distinct catalytic properties of the different oligomeric species of phosphoenolpyruvate carboxylase and changes in the equilibrium between them could be the molecular basis for an effective regulation of metabolite levels by this key enzyme of C4 plants.

The COz fixation process in C4 plants, such as maize, is initiated in the cytoplasm of leaf mesophyll cells with the p- carboxylation of phosphoendpyruvate (P-pyruvate) [l]. The reaction that takes place: P-pyruvate + HCO; + oxalo- acetate + Pi, is catalyzed by the enzyme P-pyruvate car- boxylase [2].

A divalent cation, preferentially Mg2+, is required for the catalytic activity [3]. Several cell metabolites behave as modulators of the carboxylase. Among them, malate [4, 51 and oxaloacetate [6] have been found to be inhibitors, while glucose 6-phosphate [7] and glycine [8] activate the enzyme.

Environmental factors, such as light and temperature, also affect the activity of P-pyruvate carboxylase from C4 plants. Illumination of leaves prior to extraction increases the enzyme specific activity about twofold or threefold [9 - 121. A sharp increase in the activation energy for the carboxylation of P- pyruvate has been reported to occur below 1 2 T , which was related to the cold sensitivity of maize [13].

Previous reports have shown that thiol[14- 191, histidine [20], arginine [21] and lysyl [22, 231 residues are essential for P-pyruvate carboxylase activity. Moreover, it has been reported that the incubation of P-pyruvate carboxylase from C4 plants with dithiol reductants increases the enzymic activi- ty [ l l , 17, 24, 251. Recently photoregulation of the enzyme from the same kind of plants has been reported [9 - 121. It is thought that in vivo photoregulation occurs through changes in the redox state of the carboxylase sulfhydryl groups [l 11.

P-pyruvate carboxylase from different sources has usually been described as a tetramer in its native form [2, 13, 26, 271.

Correspondence to C . S. Andreo, Centro de Estudios Fotosin- teticos y Bioquimicos, Suipacha 53 1, RA-2000 Rosario, Argentina

Abbreviation. P-pyruvate, phosphoenolpyruvate. Enzyme. Phosphoenolpyruvate carboxylase (EC 4.1 .I .31).

However, some reports, indicating changes in the state of oligomerization of the enzyme from plants exhibiting the crassulacean acid metabolism and C4 plants, suggest that dissociation processes could affect the activity in vivo [26, 28, 291.

In this paper we report on experiments which show that high ionic strength, which causes dissociation of the tetrameric form, also decreases the activity of the enzyme. The decrease of the activity seems to be related to the dissociation of the tetrameric form. The dimeric form of the enzyme reveals a specific activity one order of magnitude lower than the tetrameric form and the monomer showed almost no activity.

These results indicate that a significant part of the regula- tion of this enzyme is achieved by the associationidissociation equilibrium of its subunits. Owing to the slow conformational response of the enzyme to changes in metabolic and effector concentrations the enzyme would act as a time-dependent buffer.

MATERIALS AND METHODS

Enzyme purification and assay

P-pyruvate carboxylase was purified from maize leaves by a modification of the method of Uedan and Sugiyama [I31 described in [27] and was more than 95% pure as determined by sodium dodecyl sulphate/polyacrylamide gel electropho- resis.

Activity was measured at 30 "C by coupling the P-pyruvate carboxylase reaction to that of malic dehydrogenase and following changes in absorbance at 340 nm due to NADH oxidation in a Hitachi 150-20 spectrophotometer. The assay medium contained 50 mM TrisiHCl (pH 7 or 8), 5 mM MgCl2, 10 mM NaHC03, 0.15 mM NADH, 2 IU malic dehy-

662

drogenase, 4 mM P-pyruvate (unless otherwise indicated), and different amounts of P-pyruvate carboxylase (as indicated in the figure legends) in a total volume of 1 ml.

Size exclusion chromatography of P-pyruvate carboxylase

Gel filtration experiments were performed either on a Sepharose 6B column (600 x 15 mm) or on a TSK G 3000 SW column (570 x 9 mm) coupled to a Waters Associated Inc. HPLC system consisting of an M-6000 A chromatography pump and an U6K model valve loop injector. Equilibration and elution were performed at a flow rate of 1 ml min-' and at 25°C. The columns were calibrated with spinach coupling factor 1 (Mr 400000), catalase (Mr 240000), yeast alcohol dehydrogenase (Mr 150000), yeast hexokinase (Mr 104000), bovine serum albumin (MI 66000 monomer, and 132000 dimer) and myoglobin (MI 55000 monomer, and 110000 dimer). The void volume was determined with blue dextran. The logarithm of MI was linearly correlated with Kav. K,, is defined as K,, = (V , - Vo)/( Vt - V,) where, V, is the elution volume, V, is the void volume and V, is the volume of packed bed. Eluted protein was detected at 280 nm in a Gilson holochrome spectrophotometer.

Protein assay

using bovine serum albumin as standard. Protein concentration was determined by dye binding [30],

Materials

P-pyruvate (monocyclohexylammonium salt), NADH, pig heart malic dehydrogenase and proteins used as standards were from Sigma Chemical Co. All other reagents were of analytical grade. Solutions used for HPLC experiments were previously filtered through Millipore HA, 0.45-ym-pore-size filters, and degassed under vacuum.

RESULTS

Effect of NaCI on the quaternary structure

When P-pyruvate carboxylase was subjected to size ex- clusion chromatography on HPLC in 50 mM Tris/HCl (pH 7 or 8), a single protein peak corresponding to the 400-kDa tetrameric form was observed (Fig. 1 a and d), in accordance with previous reports [2, 131.

Fig.Ib, c, e, and f show the elution profiles of HPLC experiments run in the same buffers but in the presence of 200 mM NaCl. Fig. 1 b and 1 e shows the elution profiles obtained for samples preincubated 5 min with the elution buffer, while in Fig. 1 c and f the incubation time was 60 min. Fig. 2 shows the calibration plot of the HPLC size-exclusion column with proteins of known molecular mass. According to this calibration, we assigned apparent molecular masses to the peaks obtained in Fig. 1. Since the HPLC column revealed an exclusion limit of 300 kDa, the tetrameric form of the enzyme eluted almost in the void volume. The apparent molec- ular mass of this peak was also confirmed on a Sepharose column as previously described [27].

The elution patterns in Fig. 1 indicate that in the presence of NaCl P-pyruvate carboxylase undergoes a time-dependent dissociation into dimers and monomers at pH 8 (Fig. 1 b, c) and dimers at pH 7 (Fig. 1 e, f). The extent of dissociation was also dependent on protein concentration, increasing with

: 0 co (v

Q

400 kDo ' i @ ,II

400 kDo

200 k D o

e lut ion volume

400 kDa 4 400 kOo 200 kDo

200 kOo

elution volume

Fig. 1 . HPLC elution profiles of P-pyruvate carboxylase after gelfiltra- tion. (a, b and c) pH 8; (d, e and 9 pH 7. (a, d) Controls run in 50 mM Tris/HCI and in the absence of NaCI. (b, e) Incubated for 5 min prior to injection and eluted in the same buffer containing 200 mM NaCI. (c, 9 Preincubation was 60 min. 200 pg protein, dissolved in 200 p1 corresponding buffer, were injected in each run

protein dilution (not shown). Reinjection of the 200-kDa peak, obtained at pH 8, was followed by the appearance of 400-kDa, 200-kDa and 100-kDa peaks, indicating the exis- tence of an equilibrium between the different forms (not shown).

The process of dissociation seemed to be faster at pH 7, under these conditions the main final product was the dimeric form, even after incubation times longer than 60min. At pH 6.6 complete conversion to dimers was even faster. As is evident from Fig. 1 b, c, three different oligomeric forms were obtained at pH 8, but this equilibrium was achieved after longer periods of time or at higher salt concentrations (i.e. 400 mM NaC1). At pH values higher than 8, the equilibrium shifted even more towards the monomeric form.

In Fig.1b and c the first peak showed a small shoulder indicating either the presence of forms of higher molecular mass or, more likely, different conformations of the tetrameric form. The small peak in Fig. 1 b at higher elution volume can be assigned to the monomeric form.

When activity was measured in fractions corresponding to the different peaks, only the 400-kDa and the 200-kDa forms showed considerable activity. In the 100-kDa peak the enzyme was almost inactive.

663

501 \, \

Fig. 2. Calibration plot of the HPLC size-exclusion column from elution profiles ofproteins with known molecular mass. The individual proteins were run in 50 mM Tris/HCl pH 7 and 8 buffers in the presence of 0, 100 mM and 400 mM NaCI. Each point is the average of three individuals runs. The variation in the obtained apparent M , values was & 10% of the corresponding M,. Changes in pH (from 7 to 8) or salt concentration (0 -400 mM NaCI) did not change the apparent molecular mass of the protein standards above the obtained error limits. K,, was calculated as described in Materials and Methods from the elution volume of the proteins. The proteins used were myogiobin (O) , bovine serum albumin (H), yeast alcohol dehydrogenase (A), hexokinase (0) and CF1 (0). For the corresponding molecular masses see Materials and Methods

Effect of NaCl on enzymic activity

A time-dependent decay in the P-pyruvate carboxylase activity was observed when NaCl was present in the reaction medium (Fig. 3 a, b). At pH 8 (Fig. 3 a), reaction was started by the addition of the enzyme to a reaction medium which contained 400 mM NaCl. As shown by the first derivative trace, during the first minute the activity remained constant, decaying then to values near zero after 8 min. A similar exper- iment was carried out at pH 7 (Fig. 3 b) but in this case NaCl (200 mM final concentration) was added after reaction has been started. The change in activity was much faster than at pH 8, and reached a constant value after 1.5 - 2 min (see first derivative trace in Fig. 3 b). In the absence of salt the activity remained constant during the time required for an assay either at pH 7 or 8.

To test the response of the coupled assay system at high NaCl concentrations, 1-ml aliquots of reaction medium, con- taining all the components described above (except P-pyru- vate carboxylase), were incubated with 200 mM NaCl. At different times the enzyme was added to start the reaction. In all cases similar traces were observed. Therefore, the observed changes in activity were due to the effects of NaCl on P- pyruvate carboxylase.

From the first derivatives in Fig. 3 a and b the time course of the change in product formation as well as the initial and final reaction velocities can be obtained. At pH 8 (Fig.3a) the velocity decayed monoexponentially with a half-time of 3.4 rnin with initial and final activities of 16 U/mg and 1.2 U/ mg respectively. Whereas at pH 7 a half-time of 0.1 5 min with an initial activity of 12.5 U/mg and a final one of 2.5 U/mg were obtained.

A time-dependent decay in the activity was also observed when NaCl was replaced by Na2S04, (NH4)2S04, K2S04 or KC1 at the concentrations required to keep constant the ionic

0 .? m

-0.3 0 2 L 6 8 10

t ime (min)

-0.3 I 1

0 2 4 6 8 10

t ime (rnin)

Fig. 3. Effect of NaCl on P-pyruvate carboxylase activity. (a) The assay medium at pH 8 contained 400 mM NaCl and reaction was started by the addition of the enzyme. (b) Activity was measured at pH 7 and after 1.5 rnin 100 p1 2 M NaCl were added. In both cases 1.5 pg enzyme were used. Note: absorbance full scale of the first derivative trace was: 0.00-0.12 (pH 8) and 0.015-0.090 at pH 7

strength. For example the incubation of the enzyme with 40mM Na2S04, 40mM (NH4)2S04, 40mM KzS04 or 100 rnM KCl for 2.5 rnin in a medium of 50 mM Tris/HCl pH 8 produces an inhibition of 30-40% in the activity of the carboxylase. This inhibition is in the range of that produced by 100 mM NaCl under the same conditions.

The effect of NaCl was dependent on P-pyruvate concen- tration, provided that Mg2+ and HCO; were also present. As shown in Fig. 4, saturating P-pyruvate concentrations slowed down the loss of activity both at pH 7 and 8.

Preincubation of the enzyme in the reaction medium without Mg”, HCO; and P-pyruvate and with 200mM NaCl resulted in a decrease in the activity larger than observed for the enzyme under turnover conditions. As an example, only 54% of residual activity was retained after 2 rnin of incubation with salt at pH 8, while a sample not preincubated with NaCl but assayed with 200mM NaCl and 4mM P- pyruvate showed almost no decay at the same pH and time, as indicated in Fig.4d.

The presence of P-pyruvate or MgC12 in the incubation medium afforded no protection, indicating that only under conditions of turnover is protection observed.

Glucose 6-phosphate only partially protected the enzyme from the effect of salt (Table 1). When this activator was present during incubation at high ionic strength the activity decreased to 60% after 5 min. In contrast only about 40% of the initial activity remained in its absence. The sensitivity of P-pyruvate carboxylase to activation by glucose 6-phosphate did not change after 5 min of incubation with NaCl.

Additional experimental evidence for the above is pre- sented in Fig.5. The presence of glucose 6-phosphate abolished the effect of NaCl and, as a consequence of this,

664

0 0

a m 0 4 E, -0.025 - 0.0 50 E 5 0 - 1 0 0

-0.050 -0.100 0 1 2 0 1 2

t ime [rnin]

-0.100- -0. looL I 1 I 0 1 2 0 1 2

t ime [ rn in ]

Fig.4. Influence of substrate and salt concentration on P-pyruvate carboxylase activity. (a, b) Activity was measured with 0.5 mM P-pyruvate; (c, d) 4 mM P-pyruvate was used. Protein concentration was 1 pg ml-'. Numerals indicate NaCl concentration (mM)

Table 1. Effect of glucose 6-phosphate on NaCI-treated P-pyruvate carboxylase The enzyme (4 pg ml-') was incubated for 0 or 5 rnin in 50 mM Tris/ HCI (pH 8) plus 200 mM NaCl. After incubation, all the components of the reaction medium were added and activity was measured with 4 mM P-pyruvate. (a) No additions were made; (b) 5 mM glucose 6- phosphate was added during assay; (c) 5 mM glucose 6-phosphate was present during incubation and assay. Values in parentheses indicate percentage of activity remaining after 5 min

Time Activity

E C 0 -f m

<

a b C

min pmol min-' mg-'

0 5

11.5 15.2 15.2 4.1 (41) 5.1 (38) 9.1 (60)

the ratio of the velocity in the presence of the latter in the absence of glucose 6-phosphate increased sharply after a 2-min delay (inset of Fig. 5).

In Fig.6 the measurements of V and K , in the pH range 6.6-8 for the enzyme preincubated at pH 7 with 200 mM NaCl for 60 rnin are shown. Initial velocities were measured in media containing NaCl at the correspondent pH. At all pH values tested, linear Lineweaver-Burk plots were obtained, indicating that a single enzymatic species was present. As is evident from Fig. 6a, V showed a larger decrease with pH in the NaC1-treated sample. When V / K , was plotted against pH, similarly shaped curves were obtained in the presence and absence of NaCl, though in the presence of salt the values were lower.

0 10

t ime [ m i n l Fig. 5. Protection afforded by glucose 6-phosphate against the time- dependent decay in P-pyruvate carboxylase activity. (a) The reaction was started with 1 pg enzyme in an assay medium of pH 8 containing 400mM NaC1. (b) Similar conditions but the reaction medium contained 5 mM glucose 6-phosphate. Inset: ratio between the activi- ties in the presence and absence of glucose 6-phosphate as a function of time

DISCUSSION Previous reports concerning the P-pyruvate carboxylase

from two plants showing the crassulacean acid metabolism indicated that the enzyme could exist as a dimer or a tetramer. In one of these articles Jones et al. [26] reported a protein- concentration-dependent dissociation of the purified enzyme from Bryophyllum fedtschenkoi. Wu and Wedding found that the two different forms were related with the diurnal regula- tion of P-pyruvate carboxylase in Crassula argentea [28]. In recent papers Walker et al. presented evidence suggesting that chemical modification of thiol and imidazole groups of the

665

6.6 7.0 8 .O PH

$ ::F +NaCI

- g o 0.5

6.6 7.0 8.0 PH

Fig. 6. Kinetic parameters of NaCl-treated P-pyruvute curboxylase at different p H values. 400 pg enzyme were incubated for 60 min in 1 ml 50 mM Tris/HCl buffer pH 7 with 200 mM NaC1. Initial activity was measured in a medium similar to that described in Materials and Methods, except that the buffer was a mixture of 25 mM Hepes and 25 mM Mops, brought to pH with NaOH, and contained 200 mM NaC1. The control was incubated and measured in the absence of NaCI. K,,, and V were determined from double-reciprocal plots

carboxylase from maize induces changes in the state of oligomerization [29, 311.

In this study, gel filtration experiments revealed that ionic strength affects the oligomeric structure of the carboxylase. Protein peaks at 400 kDa, 200 kDa and 100 kDa can be cor- related with the tetrameric, dimeric and monomeric forms of the enzyme respectively. The process of dissociation was markedly affected by pH. A fast transition into dimers was induced by NaCl at pH 7. At pH 8 the dissociation, though slower, yielded also the monomer, which was the main form present after prolonged incubation at pH 8.2 or higher. It is already known that the affinity of the enzyme towards P- pyruvate [3, 201, Mg2+ [20] and effectors [5, 211 changes sharply between pH 7 and 8, and the protonation state of histidines has been proposed to be involved in such changes [3, 201. This pH-dependent transition could also be involved in the dissociation process. Walker et al. recently reported that modification of histidines with diethylpyrocarbonate pro- duced dissociation of P-pyruvate carboxylase [31].

The observed changes in the association state of P-pyru- vate caerboxylase subunits could account for the observed time-dependent decay in the activity induced by NaCl and other salts. This conclusion is supported by the similar re- sponse of the two processes to changes in pH (i. e. fast dissocia- tion into dimers and partial loss of activity at pH 7; slower and almost complete loss of activity at pH 8 and partial dis- sociation into monomers). As shown in Fig. 3 a, activity of P- pyruvate carboxylase decays to very low values after 10 min of incubation with 400 mM NaC1. This result, together with the extremely low activity measured for the 100-kDa eluted protein peak, indicate that the monomer of the carboxylase would have no activity. This conclusion is supported by the finding that the enzyme activity at pH 8 decayed mono- exponentially, although both dimer and monomer species were formed under these conditions.

The conclusion that the dimer is active is supported by the finding that after incubation under conditions where almost exclusively this form was obtained (see Figs 1 f and 6) the activity was around 50% that of the tetramer. Therefore, the pH profile shown in Fig.6 for the NaC1-treated enzyme can be attributed to the dimeric form, since it was obtained solely under these conditions. The different response of maximal velocity to NaCl at pH 7 and 8 may reflect structural changes, which would affect the pK of histidine(s) involved in the catalytic activity. The sensitivity of P-pyruvate carboxylase to high salt concentrations was enhanced when P-pyruvate was lowered to subsaturating levels. It is noteworthy that 4 mM P-pyruvate showed protection only when Mg2 + and HCO; were also present, that is to say under turnover conditions. Therefore, the substrates plus the cofactor MgZ ’ obviously increase the stability of the tetrameric form.

Interestingly, glucose 6-phosphate, which is in allosteric activator [7], revealed a protective effect against the NaCI- dependent decay in activity. In fact, after 5 min of incubation with NaCl and glucose 6-phosphate the activity was 190% higher than in a sample which lacked the activator (see Fig. 5).

Osmond and Greenway compared the salt responses of P- pyruvate carboxylase extracted from different sources, and concluded that the enzyme from leaves of C4 plants was much more sensitive to NaCl than the one extracted from the roots or that of C3 plants [32]. Similar results were obtained by Ting and Osmond [33]. However, Shomer-Ilan et al. reported that in two C, halophytes the enzyme may become salt-tolerant under turnover conditions and proposed that changes in the tertiary or quaternary structure of the protein could be the response to high salt concentrations [34].

Although the physiological significance of the effect of ionic strength on the enzyme is not clear at the present stage, P-pyruvate carboxylase may exist in an equilibrium between different oligomeric forms in the cell. This assertion would include ionic strength as another regulatory parameter to- gether with the already known pH, light and metabolites levels. This would enable us to consider P-pyruvate carboxy- lase as a hysteretic enzyme [35], i.e. an enzyme which can be efficiently regulated in its activity by association/dissociation of the subunits. In the case of P-pyruvate carboxylase, subunitlsubunit interaction (association) would increase the efficiency of the enzyme manifold, whereas dissociation (loss of subunit/subunit interaction) would decrease the enzyme activity. Moreover, since the ‘conformational response’ of the enzyme is slow compared to the catalytic turnover the enzyme can act as an efficient metabolite buffer.

This work was supported by a research grant from the Consejo Nacional de Investigaciones Cientificus y Tkcnicas (CON ICET) Argentina and by the Alexander von Humboldt Foundation, FRG. R. W. was a recipient of a Theodor Lynen Fellowship from thc Alexander von Humboldt Foundation. C. S. A. is a member of thc Career Investigator of the CONICET; D. H. G. and F. E. P. arc Fellows of the same Institution. Skillfull preparation of the figures by Mrs H. Kenneweg is very much appreciated.

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