Spinach leaf phosphoenolpyruvate carboxylase: Purification, properties, and kinetic studies

Spinach leaf phosphoenolpyruvate carboxylase: Purification, properties, and kinetic studies

ARCHIVES OF BIOCHEMISTRY Spinach AND BIOPHYSICS 163, 378-389 (1974) Leaf Phosphoenolpyruvate Properties, HENRY The Institute Pennsylvania M. M...

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ARCHIVES

OF BIOCHEMISTRY

Spinach

AND

BIOPHYSICS

163, 378-389 (1974)

Leaf Phosphoenolpyruvate Properties,

HENRY The Institute Pennsylvania

M. MIZIORK0,2

Carboxylase:

and Kinetic

THOMAS

Purification,

Studies’

NOWAK,3

AND

ALBERT

S. MILDVAN

for Cancer Research, Fox Chase Center for Cancer and Medical Sciences, Philadelphia, 19111 and The Department of Biochemistry, Uniuersity of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104 Received January

25, 1974

Spinach leaf phosphoenolpyruvate carboxylase has been purified to homogeneity using salt fractionation, chromatography, and immunologic procedures to remove contaminating ribulose diphosphate carboxylase. From gel filtration and isoelectric focusing, the molecular weight (-560,ooO) and isoelectric point (~1 = 4.9) are indistinguishable from those of ribulose diphosphate carboxylase. The subunit molecular weight of phosphoenolpyruvate carboxylase (130,000) suggests that the native enzyme is a tetramer. Kinetic studies using Mg“ or MnZ‘ as the activator indicate that the divalent cation lowers the K, of the substrate phosphoenolpyruvate by an order of magnitude and conversely, that the presence of the substrate similarly lowers the K, of the metal ion, suggesting an enzyme-metal-substrate bridge complex. Three analogs of phosphoenolpytuvate, L-phospholactate, o-phospholactate, and phosphoglycolate are potent competitive inhibitors. The inhibitor constant (K,) of L-phospholactate (2 wM) is 49-fold lower with Mn2+ as the activator than with Mg2+. An analysis of the competitive inhibition by portions of the Lphospholactate molecule (i.e., L-lactate, methyl phosphate, and phosphite) indicates this 49-fold lowering is due to increased interaction of the phosphoryl group and, to a lesser extent, of the carboxyl and C-O-P bridge oxygen of L-phospholactate with the enzyme metal complex. The results provide indirect evidence for phosphoryl coordination by the enzyme-bound divalent cation.

Phosphoenolpyruvate carboxylase [orcarboxylase thophosphate : oxalacetate (phosphorylating) EC 4.1.1.311, first described by Bandurski and Greiner (l), catalyses the divalent cation-dependent reaction of phosphoenolpyruvate (PEP)” ’ This research was supported by National Science Foundation Grant GB-27739X, and by grants to this Institute from the National Institutes of Health (CA-06927 and RR-05539), and an appropriation from the Commonwealth of Pennsylvania. z This work was begun during the tenure of a predoctoral fellowship from the National Science Foundation (H.M.M.) and a postdoctoral fellowship from the National Institutes of Health (AM-44821) (T.N.). 3 Present address: Department of Chemistry, Biochemistry, and Biophysics Program, University of Notre Dame, Notre Dame, Indiana 46556. ‘The abbreviations used are: PEP, phosphoenolpyruvate; RuP, ribulose 1,5-bisphosphate; GSH, glutathione; DTT, dithiothreitol.

with bicarbonate to form oxalacetate inorganic phosphate (Reaction 1).

and

The enzyme has been found in numerous plant and bacterial systems (2-6). Attention has been focused on the role of this enzyme in CO, fixation in plants which utilize the C,-dicarboxylic acid pathway (7). In these systems, elevated levels of PEP carboxylase make it possible for the enzyme to serve either as a primary carboxylating enzyme or as a trapping enzyme, preventing loss of CO, from the plant (8). Substrate kinetics and binding studies by Miller et al. (9) on the enzyme from peanut cotyledons have provided evidence

SPINACH

LEAF

PEP CARBOXYLASE

suggesting that a metal bridge is formed between the enzyme and PEP, in a structure homologous with that of pyruvate kinase and PEP carboxykinase. This report describes the purification to homogeneity of spinach leaf PEP carboxylase, using standard chromatographic as well as immunological procedures to permit clarification of the physical properties of this enzyme. Kinetic studies of the interaction of metal, substrates, and inhibitory analogs of PEP with the enzyme are also described. On the basis of the kinetic data, the relative importance of the carboxyl and phosphoryl groups in the binding of PEP and its interaction with the metal are discussed. EXPERIMENTAL

PROCEDURE

Materials Phosphoenolpyruvate (tricyclohexylammonium salt). NADH, dithiothreitol (DTT). and glutathione (GSH) were obtained from Sigma, Enzyme Grade Tris and (NH,),SO, from Mann. and malate dehydrogenase from Boehringer-Mannheim Corp. Salts were Baker analyzed reagent grade. Dithioglycerol was obtained from Pfaltz and Bauer (Flushing. NY). Whatman DEAE-cellulose was purchased from H. Reeve Angel, Bio-Gel A-5m and hydroxylapatite gel from BioRad, cellulose phosphate from Brown Co, (Berlin. NH), and Sephadex G-25 and G-200 from Pharmacia. Insoluble polyvinylpyrrolidone was a gift of General Aniline Film Corp. (New York, NY). Dand L-Phospholactate were prepared as described by Nowak and Mildvan (10). Phosphoglycolate was obtained from General Biochemicals (Chagrin Falls. OH). methyl phosphate from K & K Laboratories (Plainview, NY). and lactate from Pierce Chemical Co. (Rockford, IL). Ampholytes used in electrofocusing were purchased from LKB. Freund’s adjuvant. complete, was obtained from Difco Laboratories (Detroit. MI). Phosphoenolpyruvate carboxylase purified from peanut cotyledons by the procedure of Maruyama et al. (2) was generously provided by Dr. T. G. Cooper.

Methods Phosphoenolpyruuate carborylase assay. The enzyme was assayed by a variation of the coupled assay procedure of Maruyama et al. (2). The standard assay buffer contained 50 mM Tris (Cl ), pH 7.5, 10 mM KHCO,, 2 mM MnCI,, 5 mM GSH, 0.15 mM NADH, 4 mM PEP, 20 wg of malate dehydrogenase ( g 720 units per mg), and PEP carboxylase in a total volume of 1 ml. Activity was measured by following the rate of NADH oxidation, monitored at 340 nm using a

379

Gilford model 240 recording spectrophotometer. A unit of PEP carboxylase is defined as the amount of enzyme which catalyzes the carboxylation of 1 kmole of PEP per min under standard assay conditions. The reaction follows zero-order kinetics for at least 5 min and the rate is proportional to enzyme concentration over a 30.fold range to levels of 0.050 units per ml. For kinetic studies, the assay used is essentially the same as that described above, with substrate and Mn*’ or Mg9- concentrations being varied as noted. All kinetic studies were performed at 25”C, using enzyme taken through the cellulose phosphate purification step. Experiments with highly purified PEP carboxylase, free of RuP, carhoxylase, showed no detectable differences in kinetic parameters. In determining the K, of bicarbonate, solutions were prepared with freshly boiled water and kept under a nitrogen atmosphere until ready for use. In addition. the residual bicarbonate level in the assay mixture was estimated from the extent of reaction in the absence of added bicarbonate and the concentrations were corrected accordingly (2). Protein was estimated by the Lowry method (11) using ribonuclease as a standard. Preparation of antiserum to RuP, carboxylase. To complete the purification of PEP carboxylase, antibodies against contaminating ribulose diphosphate carboxylase were necessary (vide infra). New Zealand white female rabbits weighing 3-4 lb each were injected intramuscularly with homogeneous spinach leaf RuP, carboxylase. prepared as previously described (1%. 13). Two rabbits received 1 mg protein per inoculation in 1 ml of a sohition containing 10 mM phosphate buffer (pH 7.4) and 0.15 M NaCI. Three other rabbits were inoculated with 0.1 mg of protein injected as an emulsion in which the protein (in phosphate-buffered saline) had been mixed 1: 1 with Freund’s adjuvant (complete). Reinjection was performed after 2. 4, and 6 wk. Trial bleedings (5 ml) were made 4 wk after initial inoculation. Heavy bleedings (15-30 ml) were performed 3, 6. and 10 days after the last inoculation. A 142-m] quantity of serum was obtained and stored at -70°C. The specificity of the antibody was determined by the Ouchterlony diffusion technique (14). Double-diffusion plates were prepared from Iv,c agarose gel in phosphate-buffered saline. Wells, 2 mm in diameter, were cut so that a distance of 3 mm separated the edge of the center well from the edges of the surrounding wells. Undiluted serum was run overnight at room temperature against RuP, carboxylase. A single precipitant band with no spurring resulted in all cases. establishing the homogeneity of our preparation of rihulose diphosphate carboxylase (13). Polyacrylamide gel electrophoresis. Active PEP carboxylase was electrophoresed at 4°C on a 4% gel containing 12.5% ethylene glycol, 50 mhf potassium phosphate buffer, pH ‘i.5, and 1 mM EDTA. Each gel was loaded with 25 ~1 of 5 mM Na thioglycolate,

380

MIZIORKO,

NOWAK,

neutralized to pH 7.5 just prior to use, and prerun for 2% hr before loading the sample. Enzyme samples, denatured by heating for 5 min at 100°C in 8 M urea, 1% sodium dodecyl sulfate, and 5% mercaptoethanol, were applied to 6% or 10% gels containing 4 M urea and 0.1% dodecyl sulfate for electrophoresis. The buffer was 50 mM sodium phosphate buffer (pH = 7.0) with 0.1% dodecyl sulfate. After electrophoresis, gels were stained with amido black or Coomassie blue. Electrofocusing. Enzyme samples were electrofocused at 4°C in a 4-ml lo-50% Sorbitol gradient containing 16 ~1 of 40% LKB ampholytes (pH range 3-6) and 8 ~1 of 40% LKB ampholytes (pH range 6-8) per ml of gradient. After prefocusing was carried out for 2 hr, the sample was loaded and focused for 5 hr at voltages ranging from 50 V initially to 350 V finally. RESULTS Purification of PEP carboxylase. All steps were carried out at 0-5°C unless otherwise noted. The purification through the G-200 stage represents an extensive modification of the procedure of Maruyama et al. (2). Extraction. Fresh, washed spinach leaves (1500 g) were blended in 4.5 liters of 0.01 M K,HPO, containing 0.5 mM dithioglycerol and 45 g of polyvinylpyrrolidone for six 20-set intervals, each followed by a 3-min cooling period, in a Waring Blendor at medium speed. Final pH was 6.5. The mixture was strained through eight layers of cheesecloth. Ammonium sulfate fractionation. Solid (NH,),SO, was added to a level of 35% saturation. The resulting suspension was centrifuged at 10,OOOgfor 15 min and the pellet discarded Solid (NH,),SO, was added to bring the supernatant to 50% saturation. The suspension was centrifuged at 10,OOOg for 15 min and the supernatant discarded. The pellet was suspended in 20 mM potassium phosphate buffer (pH = 6.5) containing 0.5 mM dithioglycerol and 0.5 mM Na EDTA. This solution was passed through a column of insoluble polyvinylpyrrolidone (Polyclar AT) which had been preequilibrated with the same buffer, to remove plant pigments. DEAE-Cellulose Chromatography. The solution containing the partially purified enzyme was dialyzed for 4 hr with one buffer change against 100 vol of 20 mM K phosphate buffer, pH = 6.5, containing 0.5 mM dithioglycerol and 0.5 mM Na EDTA. The dialysate was mixed with a 200-ml bed volume of DEAE-cellulose which had been pretreated with acid and base, washed extensively with 10 mM Na EDTA, and equilibrated with phosphate buffer. The mixture was then poured into a column. Elution was carried out with 500-ml vol of 0.02, 0.1, 0.2, and 0.4 M K phosphate buffer (pH = 6.5), each buffer containing 0.5 mM dithioglycerol and 0.5 mM Na EDTA. Most of the enzyme activity was observed in the 0.1 M phosphate

AND

MILDVAN

eluate, with some activity found in the 0.2 M eluate. The 0.1 M eluate may be stored for several weeks at this stage at 4°C as an ammonium sulfate suspension by adding the solid salt to 50% saturation. Hydroxylapatite chromatography. Enzyme from the 0.1 M eluate of the preceding step was dialyzed for 4 hr with one buffer change against 100 vol of 10 mM K phosphate buffer, pH = 6.5, containing 0.5 mM dithioglycerol, to remove ammonium sulfate. The dialysate was applied to a hydroxylapatite column (5.0 x 6.5 cm) which had been washed and preequilibrated with 10 mM phosphate buffer. The column was eluted with 400.ml portions of 0.1, 0.2, 0.3, and 0.4 M phosphate buffer (pH = 6.5) containing 0.5 mM dithioglycerol. Activity was found in the 0.2 M and 0.3 M phosphate buffer eluates. The 0.2 M eluate, containing the major portion of the activity, was used in the next step. Most of the protein loaded on the column was ribulose diphosphate carboxylase, 95% of which was eluted by the 0.1 M K phosphate buffer, as indicated by assay and by dodecyl sulfate gel electrophoresis. However, some RuP, carboxylase adheres to the gel and elutes at the higher phosphate concentrations used to elute PEP carboxylase. Sephadex G-200 chromatography. The enzyme recovered after hydroxylapatite chromatography was concentrated by precipitation with solid ammonium sulfate and applied in a volume of 6 ml to a G-200 column (85 x 2.5 cm) which had been preequilibrated with 20 niM K phosphate buffer, pH = 6.5, containing 0.5 mM dithioglycerol, 0.5 mM Na EDTA, and 0.1 mM PEP to stabilize the enzyme. This same buffer was then used to elute the enzyme from the G-200 column. A 20.cm hydrostatic pressure head was used to increase the flow rate. The active fractions were pooled and brought to 50% saturation with solid ammonium sulfate. Cellulose phosphate chromatography. This step was necessary to remove a protein with a molecular weight of -200,000 on dodecyl sulfate gel electrophoresis. Enzyme from the preceding step was dialyzed for 4 hr with one buffer change against 100 vol of 20 mM K phosphate buffer, pH = 6.5, containing 0.5 mM Na EDTA and 0.5 mM dithiothreitol. The dialysate was loaded onto a cellulose phosphate column (14.5 x 2.5 cm) which had been preequilibrated with the same buffer. The column was washed with 160.ml aliquots of 20 mM and 100 mM K phosphate buffer, pH = 6.5, containing 0.5 mM EDTA and 0.5 mM dithiothreitol. Although protein eluted in both the 20 mM wash and in the 100 mM eluate, enzyme activity is confined to the 20 mM wash. This material is brought to 50% saturation with solid ammonium sulfate and stored at 4°C. After the cellulose phosphate step, the preparation yields four major bands on SDS gels (Fig. 1). Two bands (M, -58,000 and -14,000) correspond to subunits of residual ribulose diphosphate carboxylase

SPINACH

LEAF PEP CARBOXYLASE

FIG. 1. Polyacrylamide gel electrophoresis of PEP carboxylase in an SDS-urea system. Gels were loaded with PEP carboxylase purified through the antibody step (left) or through the cellulose phosphate step (right). Native samples were denatured, loaded on 10% I polyacrylamide gels, electrophoresed, and stained as described in Methods. Homogeneous PEP carboxylase (left) shows one band corresponding to M, = 130,000. The other bands found in the partially purified sample (right) correspond to the subunits of RuP, carboxylase (M, = 58,000 and 14,000) and some unidentified contaminants (M, = ZO,OOO-25,000).

(15). A third band contains contaminating protein in the 20,000-25,000 molecular weight range. By correlating the increase in specific activity with relative intensities of bands on dodecyl sulfate gels, the slowest moving band (M, = 130,000) was found to correspond to PEP carboxylase. A scan of the gel indicates that the purity of the PEP carboxylase after the cellulose phosphate step is approximately 25%. DEAE-cellulose chromatography. This step was necessary to remove the contaminating protein which migrated in dodecyl sulfate gel electrophoresis with a molecular weight of ZO,OOO-25,000. The ammonium sulfate suspension of PEP carboxylase after phosphocellulose chromatography is centrifuged and the pellet dissolved in a minimal volume of 20 mM

381

phosphate buffer (with 0.5 mM DTT and 0.5 mM EDTA), pH 6.5. The protein is desalted on a G-25 column (15 x 1 cm) and loaded onto a DEAE-cellulose column (11 x 1.2 cm) which had been preequilibrated with 20 mM phosphate buffer. After washing the column with 100 ml of the loading buffer, a 200-ml linear gradient (20 mM-200 mM phosphate buffer, pH 6.5) is used for elution. The 20,000- to 25,000-dalton contaminant mentioned above, with no PEP carboxylase activity, elutes at 40-80 mM phosphate. The fractions containing PEP-carboxylase activity elute at 110. to 160-mM phosphate, and are contaminated only with RuP, carboxylase, as indicated by dodecyl sulfate gel electrophoresis. Attempts to further purify PEP carboxylase by pH precipitation, specific elution from hydroxylapatite with phosphoglycolate, gel electrophoresis, and isoelectrofocusing were not successful. Gel electrophoresis at pH T.5 of a native protein sample after G-200 results in two bands. The major band (85% of the protein) contains the PEP carboxylase activity. However, dodecyl sulfate gel electrophoresis of the active band shows that ribulose diphosphate carboxylase migrates with the PEP carboxylase. Electrofocusing of the protein after cellulose phosphate purification results in a protein band centered at pH 4.9 (Fig. 2). Activity assays show that RuP, carboxylase and PEP carboxylase both focus with pl = 4.9. Samples of pure RuP, carboxylase were also electrofocused and give a pl = 4.9. Purification using immobilized antiserum to RuP, carborylase. Since conventional chromatographic, electrophoretic, and electrofocusing techniques were ineffective in removing the residual RuP, carboxylase from the PEP carboxylase preparation, antiserum to RuP, carboxylase was prepared as described in Methods. Cross-linking of the serum was accomplished by the method of Avrameas and Ternynck (16). Rabbit antiserum (28 ml) was dialyzed overnight vs 2 liters of 0.15 M NaCl containing 10 mM Na phosphate, pH 7.4. at 4°C. To 30 ml of the dialysate was added 2.8 ml of 1 M acetate buffer (pH 5.0) and the pH adjusted to 5.0 with acetic acid. A 2.5% glutaraldehyde solution (8.4 ml) was added dropwise to the stirred solution at 4°C. Upon completion of glutaraldehyde addition, the solution was allowed to warm to room temperature and solidify. After 3 hr, the gel was suspended using a Teflon-pestle homogenizer. The gel was washed with 20 mM K phosphate buffer, pH 6.5, with 0.5 mM DDT and 0.5 mM Na EDTA until no protein, as judged by the low absorbance at 280 nm, appeared in the wash. The gel was then poured into a 2.5-cm diameter column and further washed. The active fractions from gradient elution of DEAE-cellulose were loaded onto the column and the column was eluted with 20 mM phosphate buffer. The eluates containing PEP carboxylase were pooled, brought to 50% saturation with

382

MIZIORKO,

NOWAK,

AND

MILDVAN

(NHMO,, and stored frozen in liquid N,. The enzyme after this step gives one major band upon dodecyl sulfate gel electrophoresis (Fig. 1) and is free of RuP, carboxylase activity. A summary of the purification is given in Table I. The procedure results in more than a 600-fold purification, but yields less than a milligram of protein.

lar weight of which has been estimated to be 557,000 (12). Elution of the enzyme from Bio-Gel A-5M occurs between thyroglobulin (M, = 669,000) and urease (IL, = 483,000) (17). An estimate of 560,000 f 75,000 is reasonable for the native molecular weight of PEP carboxylase. ElectrophoProperties of PEP Carboxylase retie mobility on dodecyl sulfate gel sysPhysical properties and molecular tems, using markers ranging from 5,700 to weight. The elution position of PEP car- 335,000 daltons indicates a subunit molecboxylase from both Sephadex G-200 and ular weight of 130,000 * 10,000 daltons Bio-Gel A-5m coincides with that of ribu- (Fig. 3). The assumption that the native lose diphosphate carboxylase, the molecu- protein is composed of four 130,000-dalton 6,000

E a. 5,000 v v 4,0003

=O++r Y-

.-

.: 0.3 :: " 0.2

0.3 u

8 lu 0.2

El CL 0.1

0.1

0

0 IO

20

30

Tube Number FIG. 2. Density gradient isoelectrofocusing of PEP carboxylase after the cellulose phosphate step in the purification. PEP carboxylase activity (0), RuP, carboxylase activity(O), A,,, (v), and pH (A) were monitored in fractions from a lo-50% sorbitol gradient. PEP carboxylase and RuP, carboxylase migrate together, with a p1 = 4.9. Procedure is described in Methods. Temperature was 4°C. TABLE

I

PURIFICATION OF PHOSPHOENOLPYRUVATE CARBOXYLASE Stage of purification

Initial extract 35% Saturated (NH,),SO, supernatant fraction 35-50% Saturated (NH,),SO, fraction DEAE-cellulose chromatographic fraction Hydroxylapatite chromatographic fraction Sephadex G-200 fraction Cellulose phosphate chromatographic fraction DEAE-cellulose chromatographic fraction Antiserum column Purification:

Total activity (units) 465 385 407 181 134 101 59 20.4 14.9

Protein (md

Specific activity (units/mg)

RuP, carboxylase” (o/o)

15,624 10,874

0.03 0.04

-

6,940

0.06 0.07

-

2,534 128 33.9 13.8 3.16 0.75

1.05 3.0 4.3 6.5 19.9

65 zt 6 77 i 7 60 * 6 60 + 6 TJndetectable

663-fold

“The proportion spectrophotometric

of the total protein which was ribulose diphosphate carboxylase scanning at 540 nm of SDS gels stained with Coomassie blue.

was estimated

by

SPINACH

LEAF

383

PEP CARBOXYLASE

subunits would be consistent with all experimental observations. The maximal specific activity corresponds to a turnover number of 43 set’ with MnZ+ as the activator and 69 set’ with Mg2+ as the activator, assuming a molecular weight of 130,000 for the active subunit. The isoelectric points of native PEP carboxylase and RuP, carboxylase are both 4.9, as determined by electrofocusing. Stability. For prolonged storage, the enzyme is stable for at least 1 yr when frozen and stored in liquid nitrogen as an ammonium sulfate suspension. When stored in 50% ammonium sulfate at 4”C, the enzyme may be kept for 2-3 wk without appreciable loss of activity. The desalted enzyme, when kept in Tris buffer (pH 7.5) at room temperature, loses activity with a half-time of 3-4 hr. However, inclusion of 0.2 M KC1 or 0.5 M glycerol in the incubation buffer preserves 50% of the activity for at least 7 hr at room temperature. Incubation of the desalted enzyme in Tris buffer (pH 7.5) at 35°C results in a 50% decrease in activity in 40 min.

Kinetic

Studies

Activator constant of manganese and magnesium. Initial velocities were obtained as a function of Mg2+ or Mn2+ concentration at a saturating level of HCO,- (10 mM) at various levels of free PEP (Fig. 4). Concentrations of the free metal ion and free PEP were calculated, using the dissociation constant for MnPEP (1.79 x 10m3 M) (18, 19) and for Mg-PEP (5.56 x 10e3 M) (18). Because of the exceedingly weak binding of Mn’+ to HCO,(13) binary metal bicarbonate complexes could be neglected. Under conditions of saturating HCO1-, the rate equation for an enzyme, metal, substrate system is given by (20) V

v= ‘+

Kc&’ [M],[PEP],

W ( l+z+

PEP,

(2)

-4K,

where K, = ([El [MJ)/[E - M]. If rapid equilibration occurs between the enzyme, metal, and substrate prior to subsequent reaction then

K = ([El[PEPl)/F - PEPI and K,’ = ( [E - M][PEP])/[E

FIG. 3. Mobility of PEP carboxylase and marker proteins upon electrophoresis in a dodecyi sulfate-urea system. Insulin (@), myoglobin (O), chymotrypsinogen (W), ovalbumin (III), bovine serum albumin (A), phosphorylase (A), &galactosidase (v), and thyroglobulin (V) were denatured and electrophoresed as described in Methods. A plot of mobility vs subunit molecular weight is shown. From the mobility of PEP carboxylase (a), a subunit molecular weight of 130,000 daltons is estimated.

- M ~ PEP]

As previously pointed out (21), the activator constant can be determined in a double-reciprocal plot (Fig. 4) from the point at which the curves intersect, which is equivalent to extrapolating the K, of the free metal ion to a concentration of PEP equal to 0. This procedure yields K, = 400 +=60 pM for Mn2+ and K, = 667 h 50 PM for Mg2+ (Table II). Extrapolation of the K, of the free metal to infinite concentrations of PEP yields values of 37 pM for Mn2+ and 79 pM for Mg2+, indicating that this substrate significantly lowers the K, of the metal. Substrate kinetics. The kinetic data of Fig. 4 may also be plotted against the concentration of free PEP to yield the K, of PEP extrapolated to zero and infinite concentrations of the free metal (Table II). If this system showed rapid equilibrium random kinetics, the former extrapolation would yield K, and the latter would yield

384

MIZIORKO,

7 i! c

NOWAK,

AND

MILDVAN

700

z E

600

\ .c E

300

\ 9

400

2 *

300

FIG. 4. Kinetic determination of the activator constants for manganese and magnesium. Reaction mixture components included Tris-HCl, pH 7.5 (50 mM), KHCO, (10 mM), GSH (5 mM), NADH (.15 mM), malate dehydrogenase (20 rg), and PEP carboxylase (14 pg). In the Mn2+ experiment, PEP levels were: 26 WM (A), 88 f.iM (m, 351 FM (01,702 pM (a), 3511 pM (xl. In the Mg“ experiment, PEP levels were: 50 NM (A), 100 pM (v), 150 pM (O), 400 fiM (01, and 1000 pM (xl. Free metal was calculated as described in text. Final volume was 1.0 ml. Temperature was 25°C. TABLE

II

MICHAELIS CONSTANTS FOR ACTIVATORS AND SUFWRATES OF PHOSPHOENOLPYR~JVATE CARBOXYLASE

Substrate, Mn’+

cofactor

( [PEP], + 0)

& 400* 37 * 667 f 79 i

60" 2 50" 6

(PEPI,- m) Mg*+ ( PEP 1,- 0) PEP (PEPI, ( lMn’+ I,+4 401

1100 * 300

PEP (lMn’+l,([Mg*+l,+O)=)

100ii 20 250 1000

( [Mg*+I, - 30)

108 PM in the case of Mg2+. Analysis of the rates as a function of the concentration of the M-PEP complex yields linear double-reciprocal plots, which are not very sensitive to variations in the level of free metal as long as [Ml,,,, < K,. This might be expected since Eq. (2) can be rewritten: V

u-

1

1+-S-

M-PEP 108 + 40 i 10 * 110 *

10 10 3 15

/

;f

)

a

(

PF)’

t3)

s

- M - PEP])/[E where K, = ([E][M PEP] in the rapid equilibrium case (20). In most of the exneriments reported here, K, > PEP,, so that Eq. (3) is “The K, of the free divalent cation extrapolated to approximated by zero substrate concentration is defined as the activaMn-PEP Mg-PEP HCO,-

tor constant,

K, (20).

K,’ (20). Increasing

the concentration of divalent cation from zero to infinity lowers the K, of PEP from 1100 PM to 100 MM in the case of Mn2+ and from 100 PM to

V

U=

1+

K, (M - PEP)

(4)

This equation predicts competition between M, and M - PEP when M, ap-

SPINACH

proaches or exceeds K, such that the (1 + M,/K,) term is significantly greater than 1.0. While this type of behavior has been observed in an experiment at high levels of Mn*+, most of the kinetic data were collected in a region where M, < K,, explaining the insensitivity of u to variable levels of M,. The Michaelis constants for Mn PEP and Mg - PEP are listed in Table II. In the experiment where Mn2+ was observed to compete with Mn - PEP, an inhibitor constant of 460 pM for Mn2+ was determined, in agreement with the activator constant of Mn2+. These observations are consistent with the formation of a binary E - M complex without participation of the substrate, PEP. The K, of bicarbonate was determined to be 110 PM by initial velocity measurements as described in Methods at seven levels of bicarbonate ranging from 0.11 to 6.05 mM, and at saturating Mn2+ (2 mM) and PEP (4 mM). Similarly low K, values for bicarbonate have been reported for the enzyme from other plant sources (2-4) using Mg2+ as the activator. Table II summarizes the kinetic constants determined for the substrates and activators. Effect of PEP analogs on rate. The

7 E ,” :

[L-PI

I (pM)

18.6

300 -

385

LEAF PEP CARBOXYLASE

kinetic effects of phosphoglycolate and Dand L-phospholactate, which have been demonstrated to compete with PEP for several other PEP utilizing enzymes (lo), were studied with spinach leaf PEP carboxylase. Double-reciprocal plots, exemplified with L-phospholactate in Fig. 5, indicate that these compounds are linear competitive inhibitors. The phospholactates were also found to be linear competitive inhibitors in the case of the enzyme from peanut cotyledons. The inhibitor constants are summarized in Table III. In order to determine the relative contributions of the phosphoryl and carboxyl groups of phospholactate to the affinity of this analog for PEP carboxylase, the kinetic effects of methyl phosphate, phosphite, and L-lactate were investigated. These compounds also showed linear competitive inhibition with respect to PEP. Treatment of the data, analogous to that illustrated for r--phospholactate (Fig. 5), yielded the K, values listed in Table III. Effects of other compounds on rate. Because nitrate has previously been found to function as a transition state analog in the phosphoryl-transfer reaction catalyzed by creatine kinase (22), and oxalate has

L E

[L-Pi]

c :

150 -

(PM) 966

r \ .E \E

\

9.3

-

100 -

4.7

100 -

5IG E a d 0

465

50-

165

0

,050

0.10

0.15

l

.OlO

,020

0

,030

FIG. 5. Double-reciprocal plot of the initial velocity of the PEP carboxylase reaction as a function of PEP concentration, in the presence of Mn*+ (left) or Mg’+ (right) and varying concentrations of cphospholactate. Components of the 1.0.ml reaction mixture included: Tris-HCl. pH 7.5 (50 mM), K HCOJ (10 [email protected], GSH (5 mM), NADH (0.15 mM), MnCl, or MgCl, (2 mM), malate de(14 fig). Concentrations of PEP and L-phospholactate hydrogenase (20 rg), and PEP carboxylase are indicated in the figure. Temperature was 25°C.

386

MIZIORKO,

NOWAK,

been found to function in an analogous manner in enzyme-catalyzed carboxylation reactions of pyruvate (23, 24), these cornpounds were tested as inhibitors of PEP carboxylase. Oxalate is a linear competitive inhibitor with respect to PEP with an inhibitor constant of 173 PM (Table III). However, nitrate was found to inhibit noncompetitively (25) with respect to PEP. The K, values determined from the effects on slope and intercept of the double-reciprocal plots are 23 mM and 12 mM, respectively. In other experiments, a 3-fold increase of HCO,- concentration from 10 mM to 30 mM did not relieve the inhibition due to nitrate. Since the cyclohexylammonium ion has been reported to activate PEP carboxylase from E. coli (26), several monand ovalent cations (K+, Tris+, NH,‘, cyclohexylammonium+) were screened at levels up to 0.1 M and found to be without appreciable effect on the rate of the PEP carboxylase-catalyzed reaction. The tetramethylammonium cation was used in the control experiments. Similarly, acetyl CoA at a concentration of 0.25 mM, which activates the enzyme from E. coli (27), had no TABLE LINEAR

COMPETITIVE

Enzyme source

INHIBITORS

AND

MILDVAN

effect on the enzyme from spinach at 1.6 PEP. The activity of PEP carboxylase was unaffected by avidin when preincubated under conditions (20 pg/ml, 10 min at room temperature) which completely inactivated pyruvate carboxylase, indicating the absence of biotin in PEP-carboxylase (28). mM

DISCUSSION

At least 16% of the protein found in spinach leaf homogenates is ribulose diphosphate carboxylase (12). Because of the ,large excess of RuP, carboxylase over PEP carboxylase, even after most of the former enzyme is removed in the first six steps of the purification, it remains the main protein component (2 60%) of the PEP carboxylase preparation through eight of the nine steps of the purification (Table I). Due to similarities in native molecular weights of PEP carboxylase and RuP, carboxylase and due to their virtually identical mobility in gel electrophoresis or in isoelectric focusing of the native enzymes, removal of the residual RuP, carboxylase from PEP carboxylase proved to be very III

OF PHOSPHOENOLPYRUVATE PHOSPHOENOLPYRUVATE

CARBOXYLASEWITH RESPECT TO

Inhibitor

K, With Mnz+ (IlM)

Spinach leaf0

Peanut cotyledons*

L-Phospholactate P-Glycolate n-Phospholactate L-Lactate Methyl phosphate Phosphite Oxalate

7600 570 3640 173

L-Phospholactate o-Phospholactate

3.5 * 0.5 12.8 zt 0.4

2 * 0.02 6 zt 0.1 9 * 2.8 zt f i *

400 10 20 17

With Mg*+ (PM) 98 f 15 37,000 zt 10,806 4,900 f 1,300 15,700 f 1,200 -

“Initial velocity measurements were made using the standard assay system with 2 mM concentrations of ranging from 10 PM to 1000 PM, at each level of Mn2+ or Mgz+ and a minimum of four PEP concentrations, inhibitor. The highest level of inhibitor tested was 2.5-10 times higher than the K, of that compound. Temperature was 25°C. b Initial velocity experiments were made using the standard assay system except that Mn*+ concentration was 0.5 mM. A minimum of four PEP concentrations, ranging from 7 PM to 74 PM, were used at each level of inhibitor. This highest level of inhibitor tested was three to seven times higher than the K, of that compound. Temperature was 25°C.

SPINACH

LEAF PEP CARBOXYLASE

difficult. The presence of large amounts of contaminating RuP, carboxylase after the G-200 step of the preparation resulted in our misinterpretation of the number and size of subunits in PEP carboxylase as previously reported (29) and cited in a review (30). Thermal inactivation distinguished between the two activities, but since PEP carboxylase is more thermolabile than is RuP, carboxylase, this technique was of no use in further purification, Final proof that the molecules were not actually bound together was obtained only after preparing antibodies specific for RuP, carboxylase and using the immobilized antibodies to remove the last traces of this contaminant. Dodecyl sulfate gel electrophoresis of the pure PEP carboxylase indicated identical subunit,s with a molecular weight of 130,000. Hence the native enzyme from spinach, like the bacterial enzymes (31) appears to be composed of four identical subunits. Assuming 1 active site per subunit, maximal turnover numbers of 43 set-’ and 69 set’ are calculated with Mn2+ and Mg’*, respectively. Comparison of the K, of free metal (either Mn” or Mg2+) extrapolated to zero and infinite PEP indicates that PEP produces an order of magnitude tightening of the binding of metal to the enzyme. Similarly, the decrease in K, of PEP as W+)rree varies from zero to infinity suggests that the divalent cation tightens PEP binding by an order of magnitude. While these observations are consistent with formation of an enzyme-metal-substrate bridge complex, the agreement of the K, values of PEP extrapolated to infinite concentrations of either Mn2+ or Mg2+ (Table II) appears inconsistent with the 3-fold greater affinity of Mn2+ for PEP (18). Because the Michaelis constants of PEP may not represent simple dissociation constants as implied by Eq. (1) (20), the inhibitor constants of analogs of PEP, which generally represent true dissociation constants, were determined. L-Phospholactate was found to be approximately 4-fold more effective than Dphospholactate as an inhibitor of PEP

387

carboxylase from both spinach leaves and peanut cotyledons. The effect of phosphoglycolate on the spinach leaf carboxylase was intermediate. The tighter binding of the L isomer of phospholactate is similar to effects found for PEP carboxykinase and opposite to those reported for pyruvate kinase (10). The explanation for this stereoselectivity may be similar to that postulated for the case of PEP carboxykinase (lo), i.e., the methyl group of L-phospholactate may be accomodated in the bicarbonate binding site. This additional factor could result in a tighter binding constant. is The K, value for L-phospholactate 49-fold greater when measured with MgZ+ than when measured in the presence of Mn*+ (Fig. 5). To determine which portion of the phospholactate inhibitor molecule might be responsible for this difference, studies of the effect of L-lactate, methyl phosphate, and phosphite on the rate of the enzymic reaction were performed. The ratio of K, determined with Mg2* to that determined with Mn2’ is 4.9 in the case of L-lactate and 8.6 in the case of methyl phosphate. Since the ratio observed with the carboxyl-containing analog is less than that found with methyl phosphate, it might be argued that phosphate is more directly involved in formation of the enzyme-metal-L-phospholactate complex, as proposed for the substrate, PEP, by Miller et al. (9). The ratio of K, values for phosphite, which lacks the bridging oxygen atom present in methyl phosphate and L-lactate is only 4.3, suggesting a contribution by the C-O-P system to the interaction of Lphospholactate with the enzymemetal complex. Since this bridge oxygen is present in L-lactate, its ratio of 4.9 includes the contribution of this oxygen as well as the carboxyl group to the interaction of L-lactate with the enzyme-metal complex. Hence the interaction of L-phospholactate with the enzyme-bound metal appears to contain a contribution from the phosphoryl group and smaller contributions from the carboxyl and the C-O-P bridge oxygen. The order of magnitude lower K, value measured for methyl phosphate compared

388

MIZIORKO,

NOWAK,

to the K, for L-lactate further supports the importance of the phosphate group in binding at the enzyme’s active site. Direct coordination of the phosphate of phosphoglycolate by the pyruvate kinase-Mn complex has been detected by 31P nmr (32). It is of interest that the product of the K, values for L-lactate and methyl phosphate (Table III) closely approximates the K, measured for L-phospholactate in the presence of either Mn2+ or Mg’+. This agreement is probably fortuitous since the interaction of the enzyme with the hydroxyl group of lactate and the methoxy group of methyl phosphate duplicates a binding effect present in phospholactate. More typical behavior is found with phosphite where such duplication cannot occur. Thus, the product of the K, values of phosphite and L-lactate is an order of magnitude greater than the K, of L-phospholactate. This difference probably reflects the entropic advantage of the binding of L-phospholactate over the separate binding of its two components (33). The lack of potent competitive inhibition by nitrate suggests that this anion is not an analog of the transition state, and, therefore, argues against a metaphosphate intermediate as would occur in an SN1 mechanism. Indeed, metal coordination of the phosphoryl group undergoing transfer, as is suggested by the kinetic data of this paper, would inhibit an S,l mechanism and promote an SN2 mechanism (34). The noncompetitive inhibition by nitrate, with respect to PEP and HCO,- may reflect the binding of the inhibitor to a site other than the active site of the enzyme. ACKNOWLEDGMENTS We are grateful to Dr. J. P. Klinman for helpful discussions of the kinetic data, to Drs. L. H. Cohen and A. Zweidler for advice on electrophoresis, to Dr. S. Sorof for advice on immunological procedures, and to Dr. T. G. Cooper for a sample of PEP carboxylase from peanut cotyledons.

AND

MILDVAN

Biochem. Biophys. 143, 297. 4. SMITH, T. E. (1968) Arch. Biochem. Biophys. 125, 178. 5. SMITH, T. E. (1971) J. Viol. Chem. 246, 4234. 6. MAEBA, P., AND SANWAL, B. D. (1969) J. Biol.

Chem. 244, 2549. 7. HATCH, M. D., AND SLACK, C. R. (1970) Annu. Reu. Plant Physiol. 141. 8. BASSHAM, J. (1971) Proc. Nut. Acad. Ski. USA 68, 2877. 9. MILLER, R. S., MILDVAN, A. S., CHANG, H., EASTERDAY,R. L., MARUYAMA, H., AND LANE, M. D. (1968) J. Biol. Chem. 243, 6030. 10. NOWAK, T., AND MILDVAN, A. S. (1970) J. Biol.

Chem. 245, 6057. 11. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. 12. PAULSEN, J. M., AND LANE, M. D. (1966) Biochem-

istry 5, 2350. 13. MIZIORKO, H. M., AND MILDVAX, A. S. (1974) J.

Biol. Chem. 249, 2743. 14. WILLIAMS, C. A., AND CHASE, M. W. (1971) in Methods in Immunology and Immunochemistry (Williams, C. A., and Chase. H. W., eds.), Vol. III, p. 146, Academic Press, New York. 15. RUTNER, A. C. (1970) Biochem. Biophys. Res.

Commun. 39, 923. 16. AVRAMEAS, S., AND TERNYNCK, T. (1969) Immuno-

chemistry 6, 53. 17. KLOTZ, I. M., AND DARNALL, D. W. (1969) Science 166, 127. 18. WOLD, F., AND BALLOU, C. E. (1957) J. Biol. Chem. 227, 301. 19. MILDVAN, A. S., AND COHN, M. (1966) J. Biol. Chem. 241, 1178. 20. DIXON, M., AND WEBB, E. C. (1964) Enzymes, p. 439, Academic Press, New York. 21. MILDVAN, A. S., AND COHN, M. (1965) J. Biol.

Chem. 240, 238. 22. MILNER-WHITE,

E. J., AND WATTS, D. C. (1971)

Biochem. J. 122, 727. 23. MILDVAN, A. S., SCRUTTON, M. C., AND UTTER, M. F. (1966) J. Biol. Chem. 241, 3488. 24. NORTHROP, D. B., AND WOOD, H. G. (1969) J. Biol.

Chem. 244, 5820.

25. CLELAND, W. W. (1963) Biochim. Biophys. Acta 67, 173. 26. IZUI, K., NISHIKIDO, T., ISHIHARA, K., KATSUKI, H. (1970) J. Biochem. 68, 215. 27. Izur, K. (1970) J. Biochem. 68, 227. 28. GREEN, N. M. (1963) Biochem. J. 89, 599. REFERENCES 29. MIZIORKO, H., NOWAK, T., BAYER, M. E., AND MILDVAN, A. S. (1971) 162nd Nat. Meeting of 1. BANDURSKI, R. S., AND GREINER, C. M. (1953) J. the Amer. Chem. Sot., Washington, D. C., Biol. Chem. 204, 781. Biol. 50. 2. MARUYAMA, H., EASTERDAY,R. L., CHANG, H., AND 30 UTTER, M. F., AND KOLENBRANDER, H. M. (1972) LANE, M. D. (1966) J Rio/. Chem. 241, 2405. The Enzymes (Bayer, P. D., ed.), Vol. 6, p. 117, 3. MUKERJI, S. K., AND TING, I. P. (1971) Arch.

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PEP CARBOXYLASE

Academic Press, New York. 31. SUANDO, R., WAYGOOD, E. B., AND SANIYAL, B. D. (1974) J. Bid. Chem. 249, 182. 32. NOWAK, T., AND MILDVAN, A. S. (1972) Biochemistry 11, 2813.

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33. MULIVOR, R., AND RAPPAPORT,H. P. (1973) J. Mol. Biol. 76 123. 34. BENKO~IC,‘S. J., AND SCHRAY, K. G. (1973) The Enzymes (Boyer. P. D., ed.), Vol. 8, p. 201. Academic Press, New York.