The Role of Cysteine and Cystine Residues in Phosphoenolpyruvate Carboxylase from Maize Leaves

Bioehcm. Physiol. Pflanzen 1S3, 7-14 (1988) VEB Gustav Fisc-her Verlag J ena

The Role of Cysteine and Cystine Residues in Phosphoenolpyruvate Carboxylase from Maize Leaves MARIE STIBOROV A Department of Hioehemistry, Faculty of Natural Sciences, Charles University, Prague, Czechoslovakia K I] Y T e r mIn d ex: phosphoenolpyruvate carboxylase, cysteinl', half-cystine, enzyme inactivation and activation: Zea mays

Summary Phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) from maize leaves contains about thirty two half-cystine residues in the protein molecule. Eight thiol groups arc free aceessible groups. Next four thiol groups are free groups determined when the enzyme is denatured by sodium dodecylsulfate only. Next four thiol groups are available only after the treatment with dithiothreitol. Sixteen halfeystine residues are firmly bound in the enzyme protein molele. PEPC exposed to dithiothreitol or 2-mercaptoethanol l'xhibits an inerease in the enzymic activity. The enzyme is modified by p-chloromt'l'euribenzoate and Cu 2 ! ions and these modifieations result in inactivation of the enzyme. The effects of the sulfhydryl modifiers on the ehan?-,{'s of the tetrameric strnrtnre of the enzyme are examined.

Introduction

Phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) catalyzes irreversible carboxylation of phosphoenolpyruvate (PEP) to form oxalacetate in a number of organisms. In algae, bacteria and C3 plants the enzyme plays an anaplerotic role, while in C4 and Crassulacean plants it catalyzes the initial carboxylation reaction in the photosynthetic fixation of atmospheric CO 2 (O'LEARY 1982). There are indications for more than one form of PEPC in leaves (MeKERJI 1977; O'LEARY 1982; STIBOROVA and LEBLOVA 1983a). Two PEPC isoenzymes were detected in green leaves of maize (MUKERJI 1977; STIBOROVA and LEBLOVA 1983a). Major PEPC isoenzyme has more allosteric and regulation properties (MUKERJI 1977; O'LEARY 1982; STIBOROV A and LEBLOV A 1983a, 1985). This isoenzyme is studied in this paper. PEPC is a homotetramer with molecular weight of 400,000 (O'LEARY 1982; STIBOROVA and LEBLOVA 1983a; STIBOROVA et al. 1986). PEPC dissociates under certain conditions into the dimeric or monomeric forms (STIBOROV A et al. 1986). No enzyme activity was found for the monomeric or dimeric forms of the enzyme protein molecule, only the tetramer was active (STIBOROV A et al. 1986). Thiol groups playa role in the PEPC activity (HATCH and OLIVER 1978; RAGHAVENDRA and VALLEJOS 1982; STIBOROVA and LEBLOVA 1983b, 1984; IGLESIAS and ANDREO 1984a). Evidence that sulfhydryl groups are essential for the PEPC activity has been provided by studies using various sulfhydryl poisons (MANETAS and GAVALAS 1982; STIBOROVA and LEBLOVA 1983b; 1984; IGLESIAS and ANDREO 1984a). Moreover, recently IGLESIAS and ANDREO (1984 b) reported on the activation of PEPC by incubation with dithiothrcitol. MANETAS and GA YALAS (1982) suggested that the PEPC acti7

vity might be regulated in vivo by a reversible redox change of sulfhydryl groups. However, the detailed mechanisms, which explain the actual roles of thiols in this key enzyme of C4 photosynthesis are not known yet. The present paper is aimed at finding the number of half-cystine residues and the thiol groups of cysteine in the PEPC molecule and at finding the roles of these residues both in regulation of the enzyme activity and in the stabilization of the tetrameric structure of the PEPC protein molecule. Materials and Methods Plant material and chemicals PEPC was prepared from three-week old plants of maize (Zea mays L. cv. CE 205-S). Plants were cultivated at 20 to 30°C in a greenhouse under natural light. PEP from Sigma chemical Co., St. Louis, Mo, USA, Tris from Merck Darmstadt, FRG, 2-mercaptoethanol and dithiothreitol from KochLight Laboratories, Ltd., Colnbrook, England, and other chemicals from Lachema Brno, Czechoslovakia, all of analytical grade purity were used in the experiments. Isolation of the PEPC isoenzymes The PEPC isoenzymes from maize leaves were isolated close to homogeneity by the procedure reported in our earlier paper (STIBOROV A et al. 1986). The enzyme was extracted from maize leaves and precipitated by ammonium sulfate (35-55 % saturation), chromatographed on a DEAE-cellulose column, on a Sephadex G-200 column, further rechromatographed on a DEAE-cellulose culumn and on a Sepharosc 4 B eolumn. The major PEPC I isoenzyme is studied in this paper. Determination of the PEPC activity The activity was measured by the procedure reported in an earlier paper (STIBORovA and LEBLOvA 1983a). The activity was determined spectrophotometrically by monitoring NADII oxidation at 340 nm in ,l SPECORD M-40 spectrophotometer (Carl Zeiss .lena, GDR) by coupling the PEPC reaction with exogenous malate dehydrogenase or by following oxalacetate formation at 280 nm without coupling it with malate dehydrogenase. The aliquot was pipctted into a reaction mixture which contained in 1 ml: 50 mM Tris-HCl buffer pH 8.1,10 m}I NaHCO:l' 2 mM MgCI 2 , 5 mM PEP, 0.15 m}I NADH and 1 international unit of malate dehydrogenase. Oxalacctate formation was measured in the same assay medium as that deseribed above, except that NADH and malate dehydrogenase were omitted. The incubation temperature being 20°C. The following values were obta.ined in a statistical evaluation of the determination of PEPC by the above methods: x = 150.61, crn = 1.41, v % = 0.94. The values in the tables are the ayerage from five parallel experiments. Determination of sulfhydryl groups The number of free accessible sulfhydryl groups in PEPC was estimated by the method of RIDDLES et al. (1979). The reaetion of PEPC with 5,5'-dithiobis(2-nitrobenzoie) acid was started by addition of 50,ul 5,5'-dithiobis(2-nitrobenzoic) acid (10 mM in 100 mM sodium phosphate buffer pH 7.0) to 0.95 ml of purified enzyme (0.5 mg ml- 1 in 100 mM sodium phosphate pH 7.0 which contained 0.05% EDTA). The ehange in absorbance at 412 nm was recorded (SPECORD M-40 VEB Carl Zeiss JENA, GDR). The above mentioned method was also used for determination of free but nonaecessible sulfhydryl groups (groups present after denaturation of the enzyme by sodium dodeeylsulfate). In this ease 1 % sodium dodeeylsulfate was present in the reaction mixture. The number of sulfhydryl groups after reduction with dithiothreitol (10 mM) was estimated by the method of ZAHLER and CLELAND (1968). Dithiothreitol was blocked by sodium arsenite (ZAHLER and CLELAND 1968). For estimation of total half-cystine contents, the enzyme was denatured by 1 % sodium dodecylsulfate and reduced by dithiothreitol (10 mN!) with or without heating (10 min - boiling water bath) and the method described above was used (ZAHLEI{ and CLELAND 1968). The number of the total half-cystine residues was also determined by amino acid analysis after oxidation to cysteic add with pcrfonnic acid, a("('ording to the method of SCHRAM et al. (1954). An amonnt of 1mg of the enzyme was hydrolyzed for 20 or 70 h in 6 HCl at 110°C. After evaporation of the acid the amino acid content was determined in the hydrolysate on a Durrum 500 Instrument.

8

BPP 183 (1988) 1

Activation andinaciivation of PEPC The enzyme (l,uM) was incubated with dithiothreitol or 2-mercaptoethanol (10 mM) or with Cu 2+

(5 flM) or p-chloromercuribenzoate (147 p.M) in 50 mM Tris-HCI buffer, pH 7.0 at 20°C. After certain incubation period an aliquot of the mixture (0.1 ml) was transferred to the reaction medium for enzyme activity determination. Controls were run identically, except that buffer was added instead of the reagent. The samples of PEPC incubated for 30 min with the apove reagents were chromatographed on a Sepharosc 4B column (2 x 50 em) and the changes in the tctrameric structure of the enzyme were investigated. Elution was carried ont by the buffers in which the enzyme was incubated.

Results

We found 7.85 free thiol groups in the PEPC protein molecule, which were accessible to sulfhydryl reagent used in our experiments (5,5'-dithiobis(2-nitrobenzoic) acid). The number of sulfhydryl groups of PEPC was determined in the enzyme denatured by 1 % sodium dodecylsulfate, too. Under these conditions, we found 11.5 free sulfhydryl groups in the enzyme molecule. It follows from the above mentioned findings that four thiols in the enzyme molecule were hidden to sulfhydryl reagent and only after the sodium dodecylsulfate treatment they were accessible. Furthermore, the number of accessible thiol groups determined without denaturing agent (sodium dodecylsulfate) was increased by dithiothreitol (10 mM) from 7.85 to about 12.12 thiols per the PEPC protein molecule. When the enzyme was denatured by sodium dodecylsulfate and reduced by dithiothreitcl, the 16.45 thiols were found in the enzyme protein molecule. Furthermore, when the same denaturing-reducing medium was used and the samples were heated (boiling water bath), ;30.4 thiols per enzyme molecule were determined. Thus, the considerable drastic conditions must be used for estimation of all half-cystine residues (sodium dodecylsulfate, dithiothreitol, heating). The second method used for

Table 1. The content of cysteille and half-cystine residues in phosphoenolpyruvate carboxylase. Experimental conditions are described in the text (sec Ylaterial and Ylethods). Values are averages from five experiments. Reagent

Thiols per enzyme molecule

Thiols per subunit actual

nearest integer

7.85

± 0.8

1.96

2

sodium doceeylsulfate Enzyme + 10 mM

11.50

± 2.3

2.87

3

dithiothreitol Enzyme + 10 mM dithiothreitol + 1 %

12.12

± 0.2

3.03

3

sodium dodeeylsulfate (20 "C) Enzyme + 10 mM dithiothrcitol + 1 %

1GA5

± 2.8

4.11

4

sodium dodecylsulfate (100°C)

30040

7.GO

8

Enzyme (oxidation to eystei(' add and amino aeid analysis)

36.00

9.00

9

Native Enzyme Ennzyme + 1 %

± 3.8 ± 1.5

BPP 183 (1988) 1

9

estimation of total half-cystine molecules was amino acid analysis. The half-cystine residues were determined after oxidation to cysteic acid. 36.0 thiols per enzyme protein molecule were determined by the above method. The results are summarized in Table 1. The increase of the number of free thiol groups by dithiothreitol (Table 1) is correlated with the increase of the enzymic activity. The increase of the PEPC activity was also observed after the 2-mercaptoethanol treatment (Table 2). The isolated native enzyme was desalted on a Sephadex G-25 column in the Tris-HCl buffer with or without 10 mM 2-mercaptoethanol. The PEPC activity obtained by gel filtration without 2-mercaptoethanol was lower than the enzyme activity obtained with 2-mercaptoethanol (results not shown). However, when the enzyme prepared without 2-mercaptoethanol was incubated with this agent or with dithiothreitol, the PEPC activity was increased with respect to control (Table 2). The effects of sulfhydryl group modifiers on the tetrameric structure of the enzyme were studied by the chromatography on a Sepharose 4B column. The 2-mercaptoethanol-treated enzyme showed only the tetramer form (Fig. 1A). Furthermore, no changes in degree of polymerization of the enzyme during the sodium dodecylsulfate treatment was observed, too (results not shown). The incubation of the native enzyme protein with 2-mercaptoethanol (10 mM) and sodium dodecylsulfate (1 %), but without heating the samples, resulted in conversion of the tetrameric form to dimer and monomer (Fig. Table 2. The effect of p-chloromercuribenzoate, Cu 2 + ions, dithiothreitol and 2-mercaptoethanol on Thosphoenolpyrumte carboxylase. Assay conditions: The purified enzyme was desalted on a Sephadex G-25 by 25 mM Tris-HCI buffer, pH 7.0 without 2-mercaptoethanol. PEPC (1 (lM) was preincubated with reagents at 20°C. The 0.1 ml aliquots of the enzyme preincubated with reagents (see Material and Methods) were pipetted into the reaction medium which eontained in 1 m!: 50 mM Tris-HCl pH f.I, 10 m:\I NaHC0 3 , 2 mM MgC!2 and 5 mM PEP and the PEPC activity was determined. Reagent

Enzyme activity (% of control)

Time of preincubation (min)

None

100.0 100.0

120

p-chloromrfcuribenzoate (147 (lM) + PEP (1 mM) + Mg2+ (5 mM) Cn 2 + (5 (lM) + PEP (1 mM) + Mg2+ (5 m.JI) 2-mercaptocthunol (10 mM)

dithiothreitol (10 mM)

10

BPP 183 (1988) 1

14.0

73.3 14.6 34.4 93.8 33.5 100.0 IG3.5 185.8 18G.0 18G.2

30 30 30 30 30 30 30

o

30

GO

90 120

100.0

o

160.0 173.1 175.2 179.0

30 60 90

120

+

A

0.10

2'"I

:::?

005

!~

0

0 C5

I

'"

~

B ~

c:: c::, ~

'"

....... tJ

2

OlD

:>:

0

0 0.10

tJ

....

""

CIJ

2 ~

.{:)

.l3

'"tJ '"tJ

~ Cl

C

CIJ

Cl

:0 '';:::

O.OS

<.J

§

~

c... CIJ .....

0.05

~

0

0

0

1:

~ Cl

2

0./0

c:

CIJ

Cl

.c:

!}

O.OS

0

~

0

0

'f0

80

0 120

Elution volume (mlJ Fig. 1. Gel fiUration of phosphoenolpyruvate carboxylase on Sepharose 4B at various conditions. Assay conditions: All chromatograms used 50 mM Tris-Hel buffer, pH 7.0, with additions as indicated. Column was Sepharosc 4E (2 x 50 em). The enzyme was prcineubated with 10 mM 2-mercaptoethanol (30 min) (A), with 10 mM 2-mereaptoethanol and 1 % sodium dodecylsulfate at 20 DC (30 min) (E), with 10 mM 2-mercaptoethanol and 1 % sodium doderylsulfate at 100 °C (10 min) (C) and with 147 liM p-ehloromerntribenzoate (aO min) (D). The elution was carried out by the above mentioned buffers. (---) Proteinl', (0--------0) enzyme aetivity. Locations of tetra mer, dimer and monomer are indicated by arrows.

1 B). A complete transition of phosphoenolpyruvate carboxylase to monomer was achieved by heating the protein suspension which contained the enzyme, 2-mercaptoethanol and sodium dodecylsulfate (Fig. 1 C). Thus, the considerably drastic conditions must be used for total destruction of the enzymic tetrameric structure, similarly as for estimation of all half-cystine residues. PEPC is influenced by sulfhydryl poisons. The incubation of the enzyme with pchloromercuribenzoate or Cu 2 + ions resulted in inactivation of the enzymic activity (Table 2). The dissociation constants for the enzyme-Cu2+ and the enzyme-p-chloromercuribenzoate complexes were 6.uM and l00.uM, respectively. Phosphoenolpyruvate (1 mM) effectively protected inactivations due to both sulfhydryl reagent, Mg2+ (5 mM) was without this effect (Table 2). The p-chloromercuribenzoate treatment of PEPC did virtually not yield separation of the enzyme into subunits. The enzyme was mainly in the tetrameric form, but a small BPP 183 (1988) 1

11

amount of dimer was also found (Fig. 1D). The Cu 2 +-treated enzyme was eluted as a single protein peak of the tetramerie form (results not shown).

Discussion

FEPC from maize leaves studied in this paper is influenced by thiol contained reagents or by thiol poisons. Activation of PEPC by incubation with thiol contained reagents (dithiothreitol, 2-mercaptoethanol) was obtained in our experiments similarly to the results of IGLESIAS and ANDREO (1984 b). This activation is associated with the reduction of two disulfide bonds (Tables 1, 2). The changes in the redox state of above mentioned disulfide bridges in the enzyme protein molecule may be relevant to the regulation of the enzyme activity both in vitro and in vivo. The reduction of two disulfide bonds which results in the activation is not associated with the changes of the enzymic tetram eric structure. The enzyme preincubated with 2-mercaptoethanol (10 mM) at pH 7.0 was in the tetrameric form. p-Chloromercuribenzoate and Cu 2 + ions being specific thiol poisons induced inactivation of PEPC. The thiols modified by p-chloromercuribenzoate or Cu 2 + ions are probably not coupled with the maintenance of the enzyme quaternary structure. PEPC was virtually not dissociated into subunits by these agents, but the activity disappeared. Only a small amount of the dimeric form is present after the p-chloromercuribenzoate treatment (Fig. 1D). Thus, mainly free thiols are modified by thiol poisons and these groups playa role in the catalytic function of the enzyme. However, recently WALKER et al. (1986) described that p-chloromercuribenzoate partially affects the state of aggregation of the protein complex. However, the different experimental conditions were used in their experiments (WALKER et al. 1986). The content of thiol groups in PEPC was estimated by IGLESIAS and ANDREO (1984 b), who found sixteen thiol groups in PEPC. On the contrary to the finding of IGLESIAS and ANDREO (1984b), however, we found thirty two half-cystine residues. We found that sixteen half-cysteine residues which were estimated by IGLESIAS and ANDREO (1984b) arc free or partially free residues. Next sixteen half-cystine residues were found in the enzyme, which was denatured by sodium dodecylsulfate and by heating in the presence of reducing agents (dithiothreitol) (Table 2). Similarly, the thirty six halfcystine residues were determined by amino acid analysis in the enzyme, in which the disulfide bridges were oxidized to cysteic acid. The content of cysteic acid obtained by amino acid analysis was even higher than the content of half-cystine obtained by the first method. It can be explained by the method used. The cysteic acid is the first eluted from a column of amino acid analyzer before the other amino acids. Thus, the value of half-cystine content may be increased because non-characterized products of protein hydrolysis can be eluted with cysteic acid.

It can be supposed from the above mentioned results that sixteen half-cystine residues from the total half-cystine content are firmly bound in the enzyme protein molecule. The firmly bound residues can form either two disulfide bonds in eaeh monomeric subunit or disulfide bonds between subunits. Combination of both above mentioned cases can also be considered. 12

EPP 183 (1988) 1

Theoretically, the maintenance of the tetrameric form of the enzyme can be associated with disulfide bridges, with ionic, hydrophobic and hydrophilic interactions. No dissociation of PEPC was observed by the 2-mercaptoethanol treatment in the absence of sodium dodecylsulfate at pH 7.0. The similar results were obtained after the sodium dodecylsulfate treatment without 2-mercaptoethanol under the same pH. However, our previous results concerning the dissociation of the enzyme into subunits show that upon alkali (pH 9.0) or acid (pH 6.0) exposure in the presence of 2-mercaptoethanol, the enzyme partially dissociated in dimer and monomer. Alkaline pH favores depolymerization of the enzyme (STIBOROV.\ et al. 1986). The combination of denaturing and reducing agents (2-mercaptoethanol, sodium dodecylsulfate) resulted in conversion of the enzyme to dimer and monomer. Only the most drastic conditions (2-mercaptoethanol, sodium dodecylsulfate, heating) produced the total dissociation of the enzyme to the monomeric form. The conditions used for the total dissociation of the tetrameric form to monomer correlate with the conditions used for determination of the total half-cystine content. It follows from the above mentioned results that firmly bound halfcystine residues of the enzyme are accessible only after total destruction of the tetrameric form of the enzyme. Thus, it can be supposed that these firmly bound half-cystine residues can form disulfide bridges between subunits. Our work shows, however, that some other mechanisms (interactions) besides the disulfide bridges are also maintaining the integrity of the tetrameric and dimeric forms of the enzyme. Complete dissociation into monomers was achieved when the combination of all denaturing and reducing conditions was used (sodium dodecylsulfate, 2-mercaptoethanol, heating). Reducing or denaturing agents alone did not produce the total dissociation of the enzyme, but their common effects lead to total enzyme depolymerization. Recently, it was described that stability of the tetrameric form of PEPC can also be dependent, besides disulfide bridges, upon histidyl residues (WALKER et al. 1986). Thus these residues may playa role in the other mechanisms, which affects the state of the enzyme aggregation. However, the precise nature of all interactions between the subunits of the enzyme must be elucidated by further investigations. References M. D., and OLIYER, J. R.: Actiyatioll and ina(·tivation of phosphoenolpyruvate carboxylase in leaf extraets from C. speeies. Aust. J. Plant Physiol.';, 571-580(1978). IGLESIAS, A. A., and ANDHEO, C. S.: hlYolvcmcnt of thiol groups in the activity of phosphoenolpyruvate earboxylase from maize leaves. Photosynth. Res. 5, 215-226 (1984a). IGELESIAS, A. A., and ANDHEO, C. 8.: On the moleeular mechanism of maize phosphoenolpyruvate carboxylase activation by thiol compounds. Plant Physiol. 75, 983-987 (1984 b). MANETAS, A., and GWALAS, N. A.: Evideme for essential sulfhydryl group(s) in photosynthetic phosphoenolpyruvate carboxylase: protection by substrate, metalsubstrate and glueose-6-phosphate against p-chloromcfnll'ibenzoah> inhibition. Photosynthetica 16, 59-66 (1982). MUKERJI, S. K.: Corn leaf phosphoenolpyruvate earboxylascs. Purification and properties of two isoenzymps. An·h. Bioehem. I3iophys. 182, 343-351 (1977). O'I,EAHY, M. H.: Phosphoenolpyruvate earboxylase: an enzymologist's view. Annu. Rev. Plant Physiol. 33, 297-315 (1982). RAGITAYENDHA, A. S., and VALLEJOS, R. II.: Regulation of phosphoenolpyruvate carboxylase from C4 plants: Involvl'ment of thiol groups in the activity of the enzyme from Amamnthus viridis. Indian J. Expt. BioI. 20, 619-622 (1982). HATCH,

BPP 183 (1988) 1

13

RIDDLES, P. W., BLAKELEY, R. L., and ZERNER, B.: Ellman's reagent: 5,5'-dithiobis (2-nitrobenzoic) acid - a reexamination. Ana!. Biothem. 94, 75-81 (1979). 3CHRA1\1, E., MOORE, S., and BIGWOOD, E. J.: Chromatographic determination of cystine as cysteic acid. Biochcm. J. 57, 33-37 (1954). STIBOHOVj\, M., and LEBLOVJ, S.: Isolation and partial characterisation of two phosphoenolpyruvate carboxylases from maize (Zea mays L.). Photosynthetic a 17,379-385 (1983 a). STIBOROY.\, M., and LERLOV.(, S.: The role of cystcin SH groups in the phosphoenolpyruvate carboxylase molecule of maize. Physiol. Veg. 21, 935-942 (1983 b). STIROIWVA, M., and LEBLOV,\, S.: The effect of metals on maize (Zea mays) phosphoenolpyruvate carboxylase isoenzymes. In: Advances in Photosynthesis Research (SYBESMA, C., Ed.), Vo!' III. 6., pp. 473-47G, Martinus Nijhoff-Dr. Junk Publishers, The Httgue-Boston-Lancaster 1984. STIBOROVA, }f., and LEBLOV~\, S.: Aetivation of maize (Zea mays 1.) phosphoenolpyruvate carboxylase by glucose-G-phosphate and glyeine: eHeets of pH and .Jfg2+. Photosynthetica 19, 177-182 (1985). STIBOROVi\, M., LEBLovA, S., and ZBlWZEK, J.: The subunit structure and the amino aeid composition of maize (Zea mays L.) phosphoenolpyruvate earboxylase. Photosynthetica 20, 173-180 (198G). WALKER, G. R., Ku, 11. S. B., and EDWARDS, G. E.: Catalytic aetivity of maize leaf phosphoenolpyruvate carboxylase in relation to oligomerization. Plant Physio!. 80, 848-85ii (198G). ZAHLER, W. L., and CLELAXD, W. W.: A specific and sensitive assay for disulfides. J. Hio!. Cbem. 243, 716-719 (1968).

Received October 27,1986; accepted January 07,1987 Author's address: XlARIE STIBOROV A, Department of Bioehemistry, FatuIty of Natural Sciences, Charles University, Albertov 2030, 128 40 Prague 2, Czechoslovakia.

14

BPI' 183 (1988) 1