Inhibitor of C4 Acid Decarboxylases Operating in C4 ... - NCBI

Plant Physiol. ( 1987) 85, 952-957 0032-0889/87/85/0952/06/$0 1.00/0

Form of Inorganic Carbon Involved as a Product and as an Inhibitor of C4 Acid Decarboxylases Operating in C4 Photosynthesis Received for publication May 19, 1987 and in revised form July 16, 1987

COLIN L. D. JENKINS, JAMES N. BURNELL, AND MARSHALL D. HATCH* Division of Plant Industry, CSIRO, GPO Box 1600, Canberra City A.C. T. 2601 Australia ABSTRACT These studies demonstrated that CO2 rather than HC03- is the inorganic carbon metabolite produced by the C4 acid decarboxylases involved in C4 photosynthesis (chloroplast located NADP malic enzyme, mitochondrial NAD malic enzyme, and cytosolic phosphoenolpyruvate IPEP] carboxykinase). The effect of varying CO2 or HC03- as a substrate for the carboxylation reaction catalyzed by these enzymes or as inhibitors of the decarboxylation reaction was also determined. The K.1CO2 was 1.1 millimolar for NADP malic enzyme and 2.5 millimolar for PEP carboxykinase. For these two enzymes the velocity in the carboxylating direction was substantially less than for the decarboxylating direction even with CO2 concentrations at the upper end of the range of expected cellular levels. Activity of NAD malic enzyme in the carboxylating direction was undetectable. The decarboxylation reaction of all three enzymes was inhibited by added HC03-. For NADP malic enzyme CO2 was shown to be the inhibitory species but PEP carboxykinase and NAD malic enzyme were apparently inhibited about equally by CO2 and HC03-.

A key feature of C4 photosynthesis is the generation of CO2 in bundle sheath cells through the decarboxylation of C4 dicarboxylic acids. It is widely accepted that this process serves to concentrate inorganic carbon (CO2 plus HCO3 ) in bundle sheath cells and thereby largely prevent oxygenation of RuBP' and associated photorespiration (15). Radiotracer studies have provided direct evidence that relatively large inorganic carbon pools exist in the leaves of C4 plants during steady state photosynthesis (10, 1 1, 23). The composition of this pool is not known although HCO3 would be the dominant species if CO2 and HCO3 reach equilibrium at the pH in the cytosol or chloroplast stroma. However, from the model developed in the following paper it appears that this pool would consist largely of CO2 during steady state photosynthesis and that this predominance of CO2 would be vital for the proper operation of the C4 pathway (10). This conclusion depended on the assumptions that bundle sheath cells contain little or no carbonic anhydrase, that CO2 is the substrate for RuBP carboxylase (5) and that C02, as such, is the initial product of the C4 acid decarboxylation reactions in bundle sheath cells. If bicarbonate were the initial product then the proportion of total inorganic carbon present as CO2 in bundle sheath cells must always be less than the equilibrium value; this would be very low at pH values in the range from 7 to 8 (between 15 and 1.5% of total inorganic carbon; 25). Clearly, this would dramatically limit

'Abbreviations: RuBP, ribulose 1, 5-bisphosphate; PEP, phosphoenolpyruvate 952

or negate the potential physiological advantages offered by the C4 pathway. Three enzymes, NADP malic enzyme (EC 1.1.1.40), NAD malic enzyme (EC 1.1.1.39), and PEP carboxykinase (EC 4.1.1.49) have been implicated in the process of C4 acid decarboxylation in bundle sheath cells in different C4 species (9, 15). Surprisingly, evidence relating to the form of inorganic carbon involved as a product or substrate for these enzymes is either lacking or in dispute. There is clear evidence that CO2 is the substrate for PEP carboxykinase from Rhodospirillum rubrum (6) but no comparable data for the plant enzyme from any source. Dalziel and Landesborough (7) reported that wheat germ NADP malic enzyme utilizes CO2 but Asami et al. (1) concluded that HCO3- is the substrate for the maize leaf NADP malic enzyme. In the only study of the inorganic carbon substrate for NAD malic enzyme Pilone and Kunkee (24) reported that HCO3- was apparently the product formed with the Lactobacillus enzyme. During the present study we determined the nature of the inorganic carbon product formed by these three decarboxylases isolated from the leaves of C4 plants. We also determined characteristics of CO2 or HCO3- inhibition of these enzymes in the decarboxylating direction. The implications of these results in terms of the operation of the C4 pathway are considered.

MATERIALS AND METHODS Materials. NADP malic enzyme from Zea mays (modification of procedure of Hatch and Mau [ 12]), PEP carboxykinase from Urochloa panicoides (2), and NAD malic enzyme from Atriplex spongiosa (14) were prepared as previously described. Biochemicals and reagent enzymes were obtained from Boehringer-Mannheim, Australia, or Sigma Chemical Company. CO2 Solutions. Solutions of CO2 were generated by bubbling CO2 gas through distilled water kept at 0 or 10°C; at saturation the concentration of CO2 was assumed to be 70 or 50 mm, respectively (26). These solutions were kept stoppered under a CO2 gas phase and samples were removed with a syringe. Assay of Enzymes. Enzymes were assayed spectrophotometrically by following the change of absorbance at 340 nm due to oxidation or reduction of pyridine nucleotides. The temperature was either 25 or 10°C where it was necessary to follow the changes in kinetics as CO2 and HC03- were interconverted. NADP malic enzyme was assayed in the direction of malate decarboxylation in 1 ml reactions containing 25 mM TricineKOH or Hepes-KOH (pH 8.2) (or other pH as indicated), 5 mM malate, 0.5 mm NADP, and 10 mM MgCl2. For the reverse (carboxylating) direction malate and NADP were replaced by 30 mM pyruvate, 0.2 mm NADPH, and 30 mM NaHCO3 or other HCO3 or CO2 concentrations as specified.

C4 ACID DECARBOXYLASES AND C4 PHOTOSYNTHESIS

953

PEP carboxykinase was assayed in the direction of oxaloacetate decarboxylation by the procedure previously described (13). The reactions contained 50 mM Hepes-KOH (pH 7.6 or as specified), 2 mm oxaloacetate, 0.5 mm ATP, 0.5 mM MnCI2, 0.05 mM ADP, '0.6 -CA 0.2 mm NADH, 100 mm KC1, and 5 units each of pyruvate -c kinase and lactate dehydrogenase (20 units where assays were run at 10°C). For the reverse (carboxylating) reaction the above substrates were replaced by 2.5 mm PEP, 0.5 mm ADP, and 0.5 c2+c 2 mM MnC12. Malate dehydrogenase (5 units) replaced the pyru_ 0.4 vate kinase and lactate dehydrogenase. HCO+ CA 0 '1 ~ I~2 E3 NAD malic enzyme was assayed in the direction of malate N decarboxylation as previously described (16). The concentrations of reactants were varied as specified in individual experiments. HCO3 In attempts to demonstrate activity in the reverse (carboxylating) o0.2 direction the pH of 50 mm Hepes-KOH buffer was varied between 7 and 7.5 with up to 25 mM pyruvate and 15 mM HC03-. Determining the Inorganic Carbon Substrate for NAD Malic z Enzyme. Since this enzyme showed no activity in the carboxy0 lating direction the following system was used to determine the 0 1 2 nature of the inorganic carbon produced in the decarboxylating Time (min) direction. Reactions were run at 10°C to slow the conversion of CO2 to HC03-. All the reactants necessary for the decarboxylab)co 0 tion reaction were added but NAD malic enzyme was initially omitted (50 mm Hepes-KOH, pH 7.5, 0.2 mM EDTA, 5 mM ~,0.6dithiothreitol, 2 mm MnCl2, 2 mM NAD, 2 mm malate, 150 ,M c CoA). Then the following were added to provide a system for utilizing HCO3 : 4 mm PEP, PEP carboxylase (sufficient to give c2+c a maximum rate of about 0.2 umol min-' at 10°C), 5 units of malate dehydrogenase, and sufficient NADH to allow the deplet0.4tion of all residual HCO3 from the reaction mixture. Reaction Malate + NAD YpyCOr +CA components were previously largely depleted of CO2 by gassing 0, with N2 and the cuvettes were then gassed with N2 after being CA covered with parafilm. Subsequent additions were made by x 0 injection with a syringe through the parafilm and the contents 0.2 were mixed by inverting the cuvette. Lactate dehydrogenase (5 byading ethe cnetain2. M.Croi CO2 +CAO-(ia units) was also included to quantitatively convert the NADH and pyruvate formed by malic enzyme to NAD and lactate. After further addition of NADH to these reactions gave no further change of absorbance at 340 nm (i.e. HCO3- depleted and hence 2 4 0 6 no further oxaloacetate formed to oxidize NADH via malate Time (min) dehydrogenase) an excess of NADH equivalent to about 1.2 FIG. 1. Initial velocity of NADP malic enzyme (a) and PEP carboxyabsorbance units was provided. The kinetics of HCO3 production were then monitored in this system following the addition kinase (b) reactions in carboxylating direction. Reactions were initiated of C02-free NAD malic enzyme. Where indicated, 2 ,g of by adding either CO2 or HCO3 (final concentration 2.5 mm). Carbonic anhydrase (CA 1 usg) was added as indicated. Activity was measured by carbonic anhydrase was also included. Calculation of CO2 and Bicarbonate Concentrations. The con- following the change in A at 340 nm in reactions described in "Materials centration of CO2 and bicarbonate in solutions were determined and Methods." from the Henderson-Hasselbalch equation using a pKa value for bicarbonate of 6.365 at 25C or modified values at other temvolved assessing the inorganic carbon product in the dej~arboxyperatures as described by Umbreit et al. (25). lating direction in the following coupled system:

02

3

RESULTS Identification of the Inorganic Carbon Substrate. For NADP malic enzyme and PEP carboxykinase the inorganic carbon substrate was identified by following the kinetics of the reactions in the carboxylating direction initiated by the addition of either CO2 or HCO3 (Fig. 1). For both these enzymes the high initial velocity observed in reactions started with CO2 was reduced by including carbonic anhydrase. When reactions were started with HCO3 the initial velocity was low and increased over a period of 1 to 2 min. This lag was eliminated by including carbonic anhydrase in the assay mixture. We were unable to detect any significant NAD malic enzyme activity in the carboxylating direction (see next section and "Discussion"). Consequently, an alternative procedure was devised for determining the inorganic carbon substrate. This in-

Malate + NAD

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PEP + HC03-

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Oxaloacetate + NADH - Hmalate + NAD. In this system lactate dehydrogenase (LDH) was added to remove the products, pyruvate and NADH, from the NAD malic enzyme reaction. With excess of lactate dehydrogenase the pyruvate concentration would be maintained constant near zero and there would be a stoichiometric consumption of NADH. If

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Plant Physiol. Vol. 85, 1987

JENKINS ET AL.

CO2 is formed during malate decarboxylation it would be hydrated slowly by a nonenzymic reaction to give HC03- or rapidly if carbonic anhydrase (CA) is included. In the presence of PEP and PEP carboxylase (PC, this reaction uses HC03 exclusively, see Refs. 4, 22; also see Fig. 2a in this paper) the HC03 would be incorporated into oxaloacetate. In the presence of malate dehydrogenase (MDH) oxaloacetate is reduced to malate with accompanying oxidation of NADH. With an excess of these enzyrnes the system measures the rate of HCO3 formation resulting directly or indirectly from malate decarboxylation. Before commencing the assays the reaction mixtures were depleted of remaining endogenous CO2 by adding NADH until there was no further oxidation of this substrate (see "Materials and Methods"). The operation of this system as a monitor of HCO3 production was checked by comparing the responses to HC03 , C02, and C02 plus carbonic anhydrase added instead of malic enzyme (Fig. 2a). The initial rate with HC03 added was at least six times that observed with CO2. The initial rate seen in reactions started with C02 was increased to near the rate seen with HCO3 by including carbonic anhydrase. When NAD malic enzyme was added to start the reaction the rate of NADH oxidation was

m

initially slow but accelerated over a period ofmore than a minute before a steady rate was observed (Fig. 2b). Inclusion of carbonic anhydrase in the reaction mixture eliminated this lag. These results are consistent with C02 being the product of NAD malic enzyme-mediated,decarboxylation. Affinity for CO2 and V,,,, for the Carboxylating Direction. The apparent K5mO2 for the NADP malic enzyme and PEP carboxykinase was determined by varying the concentration of added HCO3 . The equivalent C02 concentrations at the particular reaction pH used were calculated from the Henderson-Hasselbalch equation. Reciprocal plots gave a KnQ2 for NADP malic enzyme of 1.1 mM (Fig. 3a). For PEP carboxykinase the KnO2 was 2.5 mm (Fig. 3b). At the likely pH of chloroplast stroma in the light of about 8.1 (17) the NADP malic enzyme was about 10 times more active in the decarboxylating direction compared with,the reverse reaction (Table I). The latter reaction contained 30 mm HCO3 (equivalent to 0.6 mM CO2 at pH 8.1). This CO2 concentration is about at the upper limit of the projected range of likely concentrations in bundle sheath cells (10, 11). At about pH 7.3 the velocities for the forward and reverse reactions were very similar. For PEP carboxykinase the velocity in the decarboxylating direction at pH 7.6 was 2.6 times that in the reverse direction (assayed with 0.5 mM C02). We were unable to detect NAD malic enzyme activity in the carboxylating direction using pH values between 7 and 7.6, pyruvate up to 25 mm, 0.15 mM NADH, and HCO3 up to 15 mm (equivalent of up to 2 mM, CO2 at pH 7). Activity was certainly less than 1% of that recorded in the decarboxylating

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