Photosynthetic Phosphoenolpyruvate Carboxylases · Photosynthetic Phosphoenolpyruvate Carboxylases...

Plant Physiol. (1973) 51, 439-447

Photosynthetic Phosphoenolpyruvate CarboxylasesCHARACTERISTICS OF ALLOENZYMES FROM LEAVES OF C3 AND C4 PLANTS'

Received for publication August 8, 1972

IRWIN P. TING AND C. B. OSMONDDepartment of Biology, University of California, Riverside, California 92502 and Department of EnvironmentalBiology, Research School of Biological Sciences, A.N. U., Canberra, 2601, Australia

ABSTRACT

A detailed comparison of green leaf phosphoenolpyruvatecarboxylases from the C4-species Atriplex spongiosa and theC3-species Atriplex hastata revealed significant physical andkinetic differences. The two alioenzymes can be separated byanion exchange chromatography but have comparable molecu-lar weights (350,000). Maximal velocity estimates were 38.0and 1.48 micromoles per minute per milligram of chlorophyllfor the carboxylases of A. spongiosa and A. hastata, respec-tively. Km phosphoenolpyruvate estimates were 0.49 and 0.08mM for the C, A. spongiosa and C3 A. hastata and the Km Mgestimates were 0.33 mM for the C4 species and 0.017 mM forthe C3 species. The activity of the phosphoenolpyruvate car-boxylase of A. spongiosa is more sensitive to chloride and phos-phate than the phosphoenolpyruvate carboxylase of A. hastata,but both are equally sensitive to Mg chelating substances suchas ATP, ADP, and citrate if assayed at their respective Km Mgvalues. A survey of the phosphoenolpyruvate carboxylases from18 C4 and C3 species resulted in mean maximal velocity esti-mates of 29.0 + 13.2 and 1.50 ± 0.57 micromoles per minuteper milligram of chlorophyll for the C4 species and C3 species,respectively. Km phosphoenolpyruvate estimates were 0.59 +

0.35 mM and 0.14 ± 0.07 mM for the C4 and C3, and Km Mgestimates were 0.50 ± 0.30 and 0.097 ± 0.057 mM for C4 andC3. All differences between means were significant at the 0.01confidence level, supporting our hypothesis that the phos-phoenolpyruvate carboxylase alloenzymes of C4 and C3 plantsare functionally different and are associated with differentphotosynthetic roles. Both function in the photosyntheticproduction of C4 acids, the phosphoenolpyruvate carboxylase ofC, species largely producing malate or aspartate (or both) asa photosynthetic intermediate and the phosphoenolpyruvatecarboxylase of C3 species producing malate or aspartate (orboth) as a photosynthetic product.

P-enolpyruvate carboxylase [orthophosphate: oxaloacetatecarboxylase (phosphorylating) EC 4.1.1.31] is an importantenzymic protein in higher plants functioning in malate syn-thesis. Malate has been known for over 20 years to be a com-ponent of photosynthesis. Bassham and co-workers (3) pro-

' This work was supported by National Science FoundationGrant GB 25878 and an Australian National University VisitingFellowship to Irwin P. Ting.

vided evidence that malate was not an intermediate of thecarbon reduction cycle, but an end product. More recently,Hatch, Slack, and co-workers (11) have shown that in certainplants (C4 plants) malate and aspartate are important inter-mediates of photosynthesis. The distinction between malate asan intermediate and as a product is most evident in pulse-chaseexperiments with "4CO,. In C4 Atriplex spongiosa malate rap-idly loses label in the chase whereas in C3 Atriplex patulamalate gains label in the chase (Osmond, unpublished). Thesetwo groups of plants, C4-plants with malate as an intermediateand C, plants with malate as a product, both synthesize malateby the coupled PEP carboxylase-malate dehydrogenase system.

Because of the distinctly different roles of malate in theleaves of C4 and C, plants, we assumed that the kinetic andphysical properties of the alloenzymes of PEP2 carboxylase inC4 and C3 species may differ. In this context, it is well knownthat the total activity of the PEP carboxylase in C, plants isgreater than in C3 plants (11). Recent data indicate that thePEP carboxylase of C4 plants is more chloride-sensitive thanthe enzyme of C3 species (22), and the C, and C, alloenzymes ofAtriplex can be separated by polyacrylamide gel electropho-resis (10). In this report, we provide evidence of clear differ-ences between the kinetic and physical properties of the PEPcarboxylase alloenzymes of C4 and C, species which may beassociated with different metabolic roles.

MATERIALS AND METHODS

Seedlings of Atriplex spp., Spinacia oleracea L., and Viciafaba L. were grown in water cultures containing 5 meq/literCa (NO3)2, 2 meq/liter KNO3, KH2PO4, and MgSO, withFeEDTA and micronutrients. Atriplex cultures were supple-mented with 5 meq/liter NaCl. All other plant materials weregrown in soil-sand cultures under the same greenhouse condi-tions. Leaves were harvested in the morning, and entire rootsystems were harvested from culture solutions.

Preparation of PEP Carboxylases. Protein was extractedfrom 30 to 50 g of Atriplex spp. leaf material by grinding twicefor 10 sec in 5 volumes of a 50 mm Bicine buffer, pH 7.8, con-taining 2 mm DTT, 2 mm EDTA, and 1% Polyclar AT. Thehomogenate was filtered through two layers of Miracloth, andcentrifuged at 15,000g for 30 min. The supernatant fluid wasbrought to 40% saturation with solid ammonium sulfate andallowed to stir gently for several hours at 4 C. The 40% am-monium sulfate preparation was centrifuged at 28,000g for30 min, and the relatively clear supernatant fluid was broughtto 55% saturation with the addition of solid ammonium sulfate

2Abbreviations: PEP: phosphoenolpyruvate; DTT: dithiothreitol;DEAE: diethylaminoethyl.

439

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Plant Physiol. Vol. 51, 1973

and stirred overnight at 4 C. The precipitate was removed bycentrifugation as above, and the pellet, containing most of thePEP carboxylase activity, was resuspended in a small volumeof 50 mm Bicine buffer, pH 7.8, for assay, or resuspended in asmall volume of 5 mm phosphate buffer, pH 7.0, for dialysisagainst the same buffer for several hours prior to DEAE-cellu-lose chromatography.The dialyzed 40 to 55% ammonium sulfate fraction was

placed on a 1.5- X 15-cm column of DEAE-cellulose (CellexD, Bio-Rad) prepared in 20 mm sodium phosphate, pH 7.0.The column was eluted with a near linear phosphate gradientfrom 0.02 M to 0.2 M as described previously (19). Fractions of7.5 ml were collected and assayed as described below. Thosewith maximal activity were pooled and again precipitated with55% ammonium sulfate. The precipitate was collected as aboveand resuspended in 50 mm Bicine buffer, pH 7.8. After dialysisagainst the same buffer, the preparation was used for kineticstudies and was stable for several days in 55% ammoniumsulfate.The 40 to 55% ammonium sulfate fraction was also sieved

through a 2.5 x 25-cm Agarose column (Bio-Rad A-0.5 M, ex-clusion limit molecular weight 500,000; 200-400 mesh). Thepreparation was put on the column with a mixture of markerenzymes (catalase, beef liver, Boehringer; assumed molecularweight 250,000; malate dehydrogenase, pig heart, Boehringer,assumed molecular weight 66,000; cytochrome c, Sigma, as-sumed molecular weight 12,500), and eluted with 50 mmBicine, pH 7.8, at a rate of 0.1 ml min-'. The eluate was col-lected in 3.5-ml fractions as described earlier for malatedehydrogenase (25).PEP carboxylase activity in the fractions was measured by

4-.

(a)a,-

1*0

0-5

lS IIIIIIIIIIIIIIIIII ~III0o0 10 20 30

FractionFIG. 1. Elution profile for the 40 to 55% saturated ammonium

sulfate fraction from DEAE-cellulose with a phosphate gradient,showing the distribution of PEP carboxylase (PC), malate dehy-drogenase (MDH), and protein (A2so) in preparations from A.hastata and A. spongiosa leaves.

coupling with added malate dehydrogenase in 10- to 100-foldexcess. The assays, containing 94 /uM NADH and the specifiedconcentrations of PEP, MgCl,, and NaHCO,, were run at30 C. In some cases, PEP carboxylase was assayed by measur-ing the appearance of oxalacetate at 270 nm or by the in-corporation of H14CO3- into malate or oxalacetate (26).

Survey of Leaf PEP Carboxylase Enzymes. The characteris-tics of PEP carboxylase activity in the extracts of leaves froma number of plants known to photosynthesize via the C3 or C,pathway were examined. Approximately 10 g of tissue werefinely chopped with a blade and extracted in 4 volumes of theabove extraction buffer by grinding twice for 10 sec in aSorvall Omnimixer. The homogenate was filtered through twolayers of Miracloth, centrifuged for 10 min at 48,000g, andimmediately assayed for PEP carboxylase (0.02-2 mm PEP)with saturating NaHCO3 (1.0 mM) and MgCl2 (3.3 mM). Maxi-mal velocity was estimated from the double reciprocal plotsobtained from these data and was expressed on a chlorophyllor fresh weight basis. Chlorophyll was determined by extrac-tion of the crude homogenate with 80% acetone and assayedat 652 nm (4).A portion of the supernatant fluid (5.0 ml) was subsequently

desalted by passage through a 1.5- X 15-cm column of Sepha-dex G-25 equilibrated with 50 mm Bicine, pH 7.8, and 2 mMDTT. Apparent Michaelis constants (Km) were estimated forPEP and Mg`+ using the desalted preparation. The Km Mg wasestimated from double reciprocal plots of l/v against 1/[S],whereas Km PEP and n number were also estimated from aplot of log vl (V-v) against log[S] (1) derived from

VSnv =

K + Sn

RESULTS

Comparison of PEP Carboxylase from Leaves of C, and C4Atriplex. The PEP carboxylase activity in the 40 to 50% am-monium sulfate fraction from extracts of A. spongiosa and A.hastata leaves showed different profiles when eluted fromDEAE-cellulose columns (Fig. 1). The PEP carboxylaseactivity of C, A. spongiosa regularly eluted ahead of the A.hastata activity, corresponding to approximately 60 and 75 mmconcentrations of phosphate, respectively. In each case, themalate dehydrogenase isoenzymes eluted as a single peakshortly after the void volume of the column. With these steepgradients, no conclusive evidence for isoenzymes of PEPcarboxylase was obtained in extracts of either plant, but theshoulder on the tail of the PEP carboxylase profile was a con-sistent feature of these experiments. The data indicate physi-cal differences between the principal PEP carboxylase proteinsof A. spongiosa and A. hastata. When these enzymes wereeluted from a Bio-Gel column, both eluted at approximatelythe same position, corresponding to a molecular weight ofabout 350,000 (Fig. 2). This molecular size estimate is con-sistent with previous observations in the literature (18, 23), andit seems unlikely that the differences between A. hastata andA. spongiosa PEP carboxylases detected by DEAE-cellulosechromatography are due to the presence of multiple units of asingle protein.The kinetic characteristics of the enzymes from C3 and C,

species prepared by DEAE-cellulose chromatography were ex-amined in detail. Figure 3 shows the initial portion of the curveof velocity versus PEP concentration for each enzyme. At lowPEP concentrations, the activity of the enzyme from A.spongiosa is distinctly sigmoidal whereas that from A. hastatais more distinctly hyperbolic. Double reciprocal plots of thesedata, however, do not adhere to Michaelis-Menten kinetics in

440 TING AND OSMOND



PHOTOSYNTHETIC PEP CARBOXYLASES

either case (Fig. 4), a feature of PEP carboxylases from higherplant leaves (19). Whether the two-phase curves of Figure 4indicate two components of the reaction is not known. Thereis a large change in the ratio of PEP and Mg over the rangeof PEP used in these experiments. The discontinuity in the

20

Fraction

cm

-

0

V

-.

FIG. 2. Elution profile for the 40 to 55% saturated ammoniumsulfate fraction from Bio-Gel A-0.5m with 50 mm Bicine showingthe distribution of PEP carboxylase activity in preparations fromA. hastata and A. spongiosa leaves in relation to marker enzymes.

A. spongiosa, Vmax=5.9

//

//

//

//

,//

i A. hastata,// Vmax= 1.1

o/o/0S o

le

,(,Ole0*05 0 1 015

PEP (mM)FIG. 3. Rate curve for PEP carboxylase from A. spongiosa (-)

and A. hastata (0) leaves at low PEP concentrations showing thesigmoidal and hyperbolic form, respectively. Assay contained 50mM Bicine buffer, pH 7.8; 3.3 mM MgCl2; and 1 mM NaHCO3.

I/v

0 5 10 15 20I

0 20 40 60t 80A hncfn+n

100

I Is llottI/SFIG. 4. Rate curve for PEP carboxylase from A. spongiosa and

A. hastata leaves over a wide range of PEP concentrations showingnonlinear double reciprocal plots. Assay conditions as in Figure 3.

double reciprocal plots does not reflect a change in rate due tothe preferential utilization of PEP or a Mg-PEP complex assubstrate. The concentrations of free PEP and Mg-PEP werecomputed at each level of added PEP from the known equilib-rium constant for the Mg-PEP complex (30), and the formof the resulting double reciprocal plot was unchanged wheneither free PEP or Mg-PEP concentration was used for calcu-lation. The data could be curvilinear and indicate enzymeactivation by PEP at high concentrations (1).

It is difficult to estimate the Km PEP from the data of Fig-ure 4, and Km estimates were therefore made from log-logplots. Using the Hill plot (1), the data of Figure 4 yield KmPEP values of 0.49 mm for A. spongiosa and 0.08 mm for A.hastata. Estimates of n from these plots were 1.25 and 0.78 forA. spongiosa and A. hastata, respectively. It was found thatrates measured in the limited range 0.1 to 2.0 mm PEP are ap-proximately linear in double reciprocal form and can be usedfor comparative purposes. All Vmax estimates were obtainedfrom double reciprocal plots with high PEP concentrations.

Kinetic data obtained with Mg as the variable resembledthose with variable PEP (Fig. 5). Estimates for Km Mg fromdouble reciprocal plots were 0.33 mm for A. spongiosa and0.017 mm for A. hastata. The rate for A. spongiosa againshowed a sigmoid tendency at low Mg concentration (Fig. 5).The Mn dependence of these enzymes was not examined norwas the pH dependence of Km PEP and Km Mg (18, 23).Michaelis constants for added NaHCO. for both PEP carbox-ylase preparations were estimated from double reciprocalplots (Fig. 6) and found to be similar for the enzyme fromboth A. spongiosa and A. hastata (0.022 and 0.018 mm, re-spectively). Since both CO, and HCO3 have been proposed asthe substrate for PEP carboxylase (7, 29), Km CO2 values of0.74 and 0.61 bLM for A. spongiosa and A. hastata were com-puted using the data of Buch (5). Slight inhibition of the A.spongiosa enzyme was noted at high NaHCO3 concentrations(Fig. 6).

0)

01)

2 0

1 5 -

V1 0-

0 5

00

Plant Physiol. Vol. 51, 1973 441




2

I/v

v

6-2 4

8, 0

0 2 4. 6 8 10I/s

05

0-4 -O

01/0o A .hastata

0

0

9

0- 1 0

60 0-2 04 06

Mg (mM)FIG. 5. Rate curve for PEP carboxylase from A. spontgiosa and A. hastata leaves as a function of Mg concentration plotted in conventional

and double reciprocal form. Assay as in Figure 3 with 1.0 mm PEP and variable MgCl2.

0.15

0.1

0

80 5I.L0

I/V 4

3

A.hastata 0 5 1011 ~~~~~I/s

0~~~~~8~~~~~~1- ~~~~5o

3

A.spongiosa 0 I/5 10/s

0 5 10

NaHCO3 (mM)FIG. 6. Rate curve for PEP carboxylase from A. spongiosa and

A. hastata leaves as a function of NaHCO3 concentration, plotted inconventional and double reciprocal form. Assay as in Figure 3, with1.0 mM PEP and variable NaHCO3.

A comparison of enzymic velocity as a function of pHrevealed slight differences between A. hastata and A. spongiosa.With HEPES buffer, enzymic velocity increased with increas-ing pH to 7.7, the highest tested. In the case of A. hastata, in-creasing pH using Bicine buffer resulted in increased velocitiesup to pH 9.5. With A. spongiosa and Bicine buffer, pH rangeabove 7.7 resulted in decreasing activity.PEP carboxylase is more sensitive to inorganic anions than

many other enzymes, and previous experiments indicated that

the enzyme from leaves of A. spongiosa was more sensitive toNaCl than that from A. hastata (22). This first report wassomewhat exaggerated by the arbitrarily chosen conditions (1mM PEP, 2 mM Mg), but the present experiments confirm thisdistinction. Whereas the A. spongiosa PEP carboxylase is moreNaCI-sensitive than the A. hastata protein, the sensitivity toNaCl is PEP-dependent (Fig. 7), and the reaction is moststrongly inhibited at or just below the Km PEP. The additionof NaCl during assay makes the A. spongiosa kinetic curvemore sigmoid in appearance (Fig. 8), whereas the A. hastatacurve remains hyperbolic (Fig. 9). n values, however, did notchange significantly. Graphical analysis shows that NaCl doesnot significantly alter the Vmax, but it increases the apparentKm PEP estimated from log-log plots (Figs. 8 and 9 and Table

1 0

A.hastata

I I A.spongiosa0 75 _

V/V0 5

0 25 _

X,

X/

/I

0 0005 002 005 02 05 1 0 2 0

PEP (mM)FIG. 7. Relative rate curve for inhibition of PEP carboxylase

from A. spongiosa and A. hastata leaves by 100 mM NaCl. Relativerate (v) in presence of NaCl is compared to rate in its absence (V)and is minimal at or near the Ko.s PEP for each enzyme.

30

20

I/v10

0

2 0

V

1.0

0 5 10 15 20I/s

0

0

_ / A.Aspongiosa0

I0

16U0 02 04 06 08

Mg (mM)1 0

0.25

0.2v

0.15

0.1

0.25

0.2

v

442 TING AND OSMOND

0

011-11"o

011-11'0

-A-

I

n I

.11




8 0

7-0 _

6-0 _

5 0

4*0

3-0

2-0

1-0

0 05 1.0

0

+1*0

-1 0

-2 0

PEP (mM ) log SFIG. 8. Rate curves for inhibition of A. sponigiosa PEP carboxylase by NaCl (eft). The data are also expressed in log-log form (right). 0:

Control; 0: 10 ms NaCI; A: 50 mm NaCl; A: 100 mM NaCl. Assay conditions as in Figure 3.

1- 0 _ o.~~08 _ / -_

0-6 I/ -n

I

06-,l 100 mM

0-4 V /e' NaCIIf

11 a'0-2 Cin

I II

0 0-5

PEP (mM)1.0

0

log SFIG. 9. Rate curves for inhibition of A. hastata PEP carboxylase by NaCl (left). The data are also expressed in log-log form (right). 0: Con-

trol; A: 100 mm NaCl. Assay conditions as in Figure 3.

I). NaCl seems, therefore, to affect the binding of PEP to theprotein, perhaps by ionic strength effects on substrate affinity.The effect can be overcome by high PEP concentrations.A similar effect was observed when phosphate buffer was

added during assay. The A. spongiosa carboxylase seemed tobe more sensitive to orthophosphate than the A. hastata pro-tein (Fig. 10). Double reciprocal graphical analyses at 100 mmphosphate and PEP concentrations from 0.1 to 1.0 mm seemedto indicate competitive kinetics with respect to PEP (Fig. 10)similar to that of NaCl (Figs. 8 and 9). These effects could bedue to a direct competition between PEP and phosphate for thereaction center or ionic strength effects as visualized for NaCl.

Effect of Metabolites and Adenine Nucleotides on Activityof Leaf PEP Carboxylases. When various substrate metaboliteswere tested at 1 mM for their effect on PEP carboxylase activityof A. spongiosa and A. hastata, only glucose-6-P activated, andcitrate and pyruvate inhibited (Table II). Citrate probably actsas a Mg chelator. The effect of pyruvate on plant PEP car-

boxylases has been reported previously (19), and inhibition by1-malate, also reported previously (14, 26), was measurablebut quite small at 1 mm. Five to 10 mm 1-malate is required for50% inhibition of both carboxylases at saturating Mg and 0.1mM PEP (unpublished observation). When 2 mm glucose-6-Pwas tested as an activator of A. spongiosa PEP carboxylase as

a function of PEP concentration, there was little effect on themaximal velocity of the reaction, but the Km PEP was low-

Table I. Effect of NaCI oni PEP Carboxylases of A. hastataand A. sponigiosa

Vmax values were estimated from double reciprocal plots andexpressed relative to 0 mm NaCl. Km and ni values were estimatedfrom log-log plots shown in Figures 7 and 8.

NaCI (mm)

0 10 50 100

A. sponigiosaVmax 1.00 0.98 0.98 0.93Km 0.49 0.8 1.21 2.01i 1.25 1.22 1.25 1.21

A. hastataVmax 1.00 ... ... 1.07Km 0.08 ... ... 0.275n 0.78 ... ... 0.76

ered. Log-log plots of these data resulted in a Km PEP estimateof 0.46 mm (n = 1.10) and 0.12 mm (n = 1.2) for the absenceand presence of glucose-6-P, respectively. The activating effectof glucose-6-P was also tested with PEP carboxylase preparedfrom maize and sorghum with substantially the same results;i.e., glucose-6-P decreased the apparent Km PEP, with rela-tively little effect on the Vmax or the n value.

0-~~~~~~/o,- 10mM

ox/</ NaC

/ ~~A

50mM

A

° /^/ a~~-1 loo0 mm'L~~~ , .~

+1 0

0-Io.I

.0-

-2 0 OZ 1O0,^ +10

/gX 100 mM0 - NaC

°/ L'-'

- -1 00 II

443Plant Physiol. Vol. 51, 1973




100 200

Pi (mM)

80 -

A.spongiosa o

60 -

I/V Pi 1OCmM /40 -

020 -

o < -L- ,I

60 - A. hastata

I/v.0- P cmPi 100mM

<

0

,l 0

-5 10

I/ [PEP]

FIG. 10. Relative rate curves for inhibition of PEP carboxylase from A. sponigiosa and A. hlastata leaves by inorganic phosphate (left). Therate curves are also expressed in double reciprocal form (right). Assay conditions as in Figure 3, with 0.1 mm PEP.

Table II. Effect of Various Metabolites oni PEP CarboxylaseActivity

Activities were estimated at approximate Km substrate values:A. sponigiosa, PEP, Mg2+ = 0.5 mM; A. hastata, PEP, Mg2+ = 0.1mM.

A/AolAletabolite (1 mm)

A. Izasiata A. spongiosa

Control 1.0 1.0Glucose-6-P 1.22 1.7Fructose-6-P 0.94 1.0Glucose-i-P 1.0 1.08Fructose-1, 6-diP 0.9 0.82Ribose-5-P 0.94 0.96Ribulose-1, 5-diP 1.21 0.94Glycerate-3-P 0.89 0.94Gluconate-6-P 0.971-Malate 0.94 0.811-Aspartate 0.94 0.93Citrate 0.45 0.43Pyruvate 0.63 0.92

l Ratio of initial rate in presence of metabolite to initial rate inabsence of metabolite.

A study of the interaction between glucose-6-P and chloridesuggested independent effects. Glucose-6-P decreases the KmPEP with no effect on the maximal velocity, and chloride in-creases the Km PEP with no apparent effect on the maximalvelocity. Interaction between glucose-6-P and NaCl was paral-lel at glucose-6-P concentrations from 0.1 to 10 mM andchloride from 5 to 200 mM when assayed at the Km PEP. Agiven concentration of chloride resulted in a proportionate de-crease in rate regardless of the glucose-6-P concentration, anda given concentration of glucose-6-P increased the rate by aconstant proportion, regardless of the chloride concentration.Although ribulose-1,5-diP enhanced the PEP carboxylase ac-tivity of A. hastata (Table II), the effect was not consistent andwas not investigated in detail.

Adenine nucleotides were tested for their effects on thereaction rates of A. hastata and A. spongiosa PEP carboxyl-ases. At saturating PEP and Mg concentrations, neither en-

zyme was markedly affected by 1 mm ATP, ADP, or AMP(Table III). At saturating PEP concentrations and a Mg con-centration approximating the Km Mg, ATP inhibited bothproteins 86 to 90%, ADP inhibited 50 to 65%, and AMP hadvirtually no effect. At saturating Mg and PEP concentrationsapproximating the Kmn PEP, ATP and ADP had slight inhibi-tory effects. Although the A. spongiosa protein appears to bemore sensitive to ATP and ADP than the A. hastata protein(Fig. 11), when assayed at their respective Km Mg, smalldifferences were noted (Table III). The data suggest that theadenine nucleotide inhibition is probably by way of chelationof Mg.

Comparison of PEP Carboxylases from a Variety of C3 andC, Plants. The data obtained from a survey of PEP carboxylaseactivity from green, photosynthetic leaves of a variety of plantsagreed with the A. spongiosa and A. hastata comparison (TableIV). In general, the data presented in Table IV indicate thatthe maximal velocities, when measured at pH 7.8 in a fresh

Table III. Effect of Adeniine Nucleotides ont PEPCarboxylase Activity

Saturating PEP and Mg for A. spongiosa were 4 and 8.2 mM;for A. hastata saturating PEP and Mg were 2 and 4.1 mm. Dataare expressed as a ratio of initial rate in presence of adeninenucleotide to initial rate in absence of nucleotide.

A/AoNucleotide and Conditions of Assay

A. spongiosa A. hastala

1 mM ATPWith saturating PEP, Mg 0.93 0.9With saturating PEP, Km Mg 0.14 0.116With saturating Mg, Km PEP 0.76 0.97

1 mM ADPWith saturating PEP, Mg 1.0 1.0With saturating PEP, Km Mg 0.52 0.36With saturating Mg, Km PEP 0.84 0.79

1 mM AMPWith saturating PEP, Mg 0.95 1.07With saturating PEP, Km Mg 1.07 0.97With saturating Mg, Km PEP 0.93 1.0

- Aha

A has

V/V0 5

,tata

A. spongiosa

100

444 TING AND OSMOND




Oh

Os

03

v

02

0.1

0 05 1.0 15 20 C

0~~~~~~~

0~~~~

I a/ ~~~A.spongiosa

0

/A

/

La^I'I--0.5 1.0 1S 20

Mg (mM)FIG. 11. Rate curves for Mg dependence of PEP carboxylase with (A) and without (0) 1 mm ATP. A. hastata extracts assayed as in Figure

3 with 1 mm PEP, A. spongiosa extracts with 2 mt PEP.

Table IV. Kinetic Comparisoni of P-enolpyruvate Carboxylases from Leaves of Cs and C4 Plants

Vmax determined in fresh homogenates by extrapolation of double reciprocal plots with variable PEP concentration and saturatingMg and NaHCO3. Km Mg from double reciprocal plots, Knm PEP and it from log-log plots. All differences between means significantat 0.01 level.

Species and Photosynthetic Type' Vmax Km Mg Km PEP n

j&moles/min *g fr. wet. moles/min-mg chl mm mM

C4Panticum maximum Jacq. 53.5 37.6 0.70 0.53 1.06P. miliaceum L. 12.1 13.0 0.95 1.40 1.10Saccharum officinarum L. 24.2 50.0 1.00 0.80 1.03Chloris gayana Kunth. 13.4 15.6 0.14 0.73 1.13SorghumbicolorL. 17.0 25.6 0.50 0.63 1.12Zea mays L. 19.2 12.9 0.46 0.35 1.00Atriplex spongiosa F.v.M. 16.1 38.0 0.33 0.49 1.25A. niummularia Lindl. 9.7 39.6 0.50 0.63 1.12Amaranithus edulis Speg. 41.0 38.5 0.21 0.19 1.17A. palmeri Wats. 18.3 19.2 0.18 0.19 1.34X(C4) S.E. 22.5 4 14.0 29.0 + 13.2 0.50 ± 0.30 0.59 + 0.35 1.13 + 0.1

CsSpinacea oleracea L. 1.6 1.54 ... 0.13 0.86Vicia faba L. 1.68 1.63 0.095 0.11 0.94Phaseolus vulgaris L. 1.47 1.52 0.128 0.30 0.93Nicotiana glutinosa L. 2.52 2.72 ... 0.08 0.90Gossipium hirsutum L. 1.62 1.15 0.180 0.18 0.85Helianthus annuus L. 1.01 1.10 0.11 0.11 0.84Atriplex hastata L. 1.48 1.48 0.017 0.08 0.78Triticum vulgare Vill. 0.66 0.82 0.053 0.09 0.92X(C3) i S.E. 1.51 0.54 1.50 ± 0.57 0.097 :+ 0.057 0.14 ± 0.07 0.88 4 0.05

t[X(C4) - X(C3)] 4.76 6.55 4.17 4.03 7.95

1 X = mean value; t[X(C4) -X(Cz)] = Student's t value for difference between mean.

homogenate, are on the average 15 or 20 times greater in theextracts of leaves of C4 plants when expressed as a function offresh weight or chlorophyll. Furthermore, the Michaelis con-stants for Mg and PEP are significantly greater (4-5 fold) forthe C4 leaf alloenzyme than for the C3 leaf alloenzyme. ThePEP carboxylases from leaves of C, plants had n values greater

than unity whereas those from leaves of C, plants had n valuesless than unity.

Although the variation about the means is great, differencesbetween the means are significant at the 0.01 level in eachcase. The data suggest that there is large variation of Vm..values and Km values among all plants, but that on the average

Plant Physiol. -Vol. 51, 1973 445




the Vmax and Kmn for Mg and PEP are significantly greater forthe PEP carboxylases of C4 leaves.

DISCUSSION

The data presented here indicate major differences betweenthe PEP carboxylase alloenzymes in leaves of plants which fixCO, via different photosynthetic pathways. As is well known,the maximal velocity of leaf PEP carboxylase in plants withthe C4 pathway of photosynthesis is 15- to 20-fold greater (on achlorophyll or fresh weight base) than that in leaves of C3plants in which photosynthetic CO2 fixation involves the carbonreduction cycle alone (11). In closely related C. and C4 species,the PEP carboxylase alloenzymes have clearly distinct chro-matographic and electrophoretic profiles (10) and, althoughapparently similar in molecular weight, show quite distinctproperties with respect to apparent Michaelis constants forPEP and Mg (but not for CO-, HCO,-) and with respect to in-organic anion inhibition (22).

In a survey of C3 and C, plants, the Kmn PEP and Km Mgvalues were significantly greater for the alloenzyme in C4 spe-cies, which was also more sensitive to inorganic anion inhibi-tion. The Kit PEP values obtained extend over the range re-ported for this enzyme isolated from nongreen tissues (23, 26).Although cotton leaf PEP carboxylases show Kin PEP andKm Mg comparable to the other C, plants examined here (19),our values obtained for Spinacia are considerably lower thanthose reported earlier (2).The properties of photosynthetic PEP carboxylases in rela-

tion to the substrate PEP are of particular interest. Data ofenzymic rate versus PEP concentration do not obey simpleMichaelis-Menten kinetics and appear as a two-phase or per-haps a single curvilinear function in double reciprocal formas found for the enzyme in cotton leaves. It is difficult toascribe these observations to artifacts arising from the coupledassay system from the interaction of Mg and PEP, from acontaminating PEP phosphatase, or from the instability ofPEP in dilute solution. A linear fit to the log-log Hill plot (1)was obtained with respect to PEP for both C, and C4 alloen-zymes of PEP carboxylase, yielding n values characteristicallygreater than 1 for the C4 alloenzyme (1.13 + 0.01) and lessthan 1 for the C. alloenzyme (0.88 + 0.05). Although nomechanistic significance is here attached to the n values andthe variation from unity (1, 24), they may indicate importantand distinctive features of the enzymic reaction rate. Thus thehigher n value for the PEP carboxylase alloenzyme of C4 spe-cies stems from the sigmoid shape of the rate curve and indi-cates a slower than anticipated reaction at low substrate con-centrations. The n value, considered with higher Km PEP andhigher Vmax estimates, suggests that the alloenzyme in leaves ofC4 plants is a more efficient catalyst at high PEP concentrationsand that the alloenzyme in leaves of C, plants is more efficientat low PEP concentrations.The size and kinetics of PEP pool changes during photosyn-

thetic transients in C3 and C4 species have not been compared.Certainly, PEP pools behave as classical CO, acceptors whenCO2 is removed during steady state photosynthesis in maizeleaves (9). Contradictory results are obtained with CO2 im-pulses to illuminated maize leaves maintained in CO2-free air(15), but in both experiments the PEP pools are clearly inequilibrium with those of other C, compounds. The generationof PEP as a photosynthetic substrate differs in leaves of C, andC4 plants. Pyruvate, P, dikinase, a light-activated enzyme, ap-pears to be responsible for the direct regeneration of PEP frompyruvate in leaves of C4 plants (12). This enzyme is scarcelydetectable in C, plants, and PEP is presumably derived from3-PGA via phosphoglyceryl mutase and enolase (3). The leaves

of green plants fix CO, in the dark via PEP carboxylase, pre-sumably consuming glycolytic PEP in the process. Availabilityof substrate must be an important factor in maintaining theslow rate of dark CO2 fixation in leaves of C4 plants. Our un-published data show that leaf slices of A. spongiosa and A.hastata fix CO2 in the light under optimal conditions at 3.9 and3.0 j,moles/min mg chl, respectively. In the dark the corre-sponding values are 0.05 and 0.03 ,tmoles/min mg chl.Clearly, the differences in PEP carboxylase activity in C4 andC, leaves are not expressed during dark CO, fixation. Thecombination of a high Kin PEP; a substantial reduction inPEP pools in the dark, (9) possibly due to inactivation ofpyruvate, P, dikinase, and the somewhat sigmoid rate kineticsof the PEP carboxylase of C4 leaves evidently contribute to theslow rate of dark CO2 fixation in these plants. Leaves of C4plants and Crassulacean acid metabolism plants, however, havecomparable PEP carboxylase activities (14) and presumablydraw on glycolytic PEP during dark CO, fixation. For reasonswhich are not readily apparent, Crassulacean acid metabolismplants fix CO2 very much more rapidly in the dark than doleaves of C4 plants (27).The sigmoidal nature of the rate curves for the PEP carbox-

ylase alloenzyme from leaves of C4 plants indicates it may bean allosteric protein, similar to the same enzyme isolated fromthe Enterobacteriaceae (6). The latter PEP carboxylase is in-hibited by malate (20) and aspartate (17) and is activated byacetyl-CoA (6), fatty acids (13), and fructose-1 ,6-diP (8).Although plant PEP carboxylases are inhibited by malate (14,25), oxalacetate (16, 26), and other organic acids (19), concen-trations approaching 10 rim are required for 50% inhibition.Compounds which chelate Mg inhibit both alloenzymes ofPEP carboxylase from leaves of C, and C4 plants. For exam-ple, EDTA, citrate, ATP, and ADP were inhibitory, but AMPwas not. A stimulatory effect of glucose-6-P was noted; detailswill be published elsewhere. The glucose-6-P activation wasparticularly effective at low PEP concentrations and wasmore pronounced with the alloenzyme from C4 A. spongiosa.The physiological significance is difficult to ascertain, however.In the simplest sense, we visualize some degree of product andprecursor control of CO2 fixation via PEP carboxylase at thesubstrate level by the above compounds. Accumulation ofphotosynthate may regulate carbon flow of photosynthesis,and the accumulation of glucose-6-P could, for example, stim-ulate CO2 fixation via PEP carboxylase.The above-mentioned properties clearly distinguish the

PEP carboxylase alloenzymes of leaves of C, and C4 plants.These enzymes are associated with different photosyntheticfunctions: the generation of photosynthetic intermediates ofthe C, pathway and the generation of photosynthetic productsfrom the carbon reduction cycle in C, plants. It remains tobe seen whether these enzymes function in the nonphotosyn-thetic pathways of CO2 fixation in green leaves. The latterpathway functions in A. spotngiosa leaves in the dark, for ex-ample, to provide organic anions for ionic balance in leaf cellvacuoles (21), the process being analogous to that in root cells.Published evidence (26) and that presented in the followingpaper (28) suggest that a further distinct alloenzyme of PEPcarboxylase may be associated with nonautotrophic CO2 fixa-tion in nongreen tissues of C, and C, plants.

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