NADP-malic enzyme from maize leaf: Regulatory properties

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 196, No. 2, September, pp. 581-587, 1979

NADP-Malic

Enzyme from Maize Leaf: Regulatory

Properties]

S. ASAMP, K. INOUE, AND T. AKAZAWA Research Institute

for Biochemical Regulation, School of Agriculture, Chikuea, Nagoya 464, Japan

Nagoya

University,

Received February 5, 19’79;revised April 26, 1979 The regulatory properties of purified maize leaf NADP-malic enzyme (EC 1.1.1.40) were studied at three different pHs and the following results were obtained. (a) At pH 7.5 enzyme activity reaches a maximum at 0.4-0.8 mM malate depending on the MgZ+concentration, and higher levels of malate result in marked substrate inhibition; with increasing pH the degree of substrate inhibition is reduced to where at pH 8.4 little or no inhibition is observed. (b) The inhibitory effect of malate is more pronounced at 1 mM Mg*+ than at 5-10 mM Mg*+ in the pH range of 7.5 to 8.4; a plot of enzyme activity vs MgZ+ concentration at 3 mM malate follows Michaelis-Menten kinetics at both pH 7.5 and 8.4; the apparent affinity of the enzyme for Mge+ at pH 8.4 was threefold greater than that at pH 7.5. (c) The activity of NADP-malic enzyme decreases as the ratio of NADPH/NADP increases, and this effect is enhanced at lower pH. (d) Various a-keto acids including glyoxylate, oxaloacetate, and cY-ketoglutarate inhibit NADP-malic enzyme activity, whereas HCOI-, pyruvate, and other organic acids, sugar phosphates, and amino acids have little or no effect on the activity of the enzyme. Based on these experimental findings, the regulatory properties of maize leaf NADP-malic enzyme are discussed with respect to its key role in net CO* fixation in maize bundle sheath chloroplasts during C, photosynthesis.

In leaf tissue of maize (Zea mays L.), a representative NADP-malic enzyme-type C, plant, the major photosynthetic product of the mesophyll, malate is transported to the bundle sheath chloroplasts and oxidatively decarboxylated by NADP-malic enzyme (EC 1.1.1.40); the HCO,- released (cf. (1)) is refixed by RuPz3carboxylase (EC 4.1.1.39) (2-4). It has been reported that in NADP-malic enzyme-type Cqplants, the ac-

tivity of this decarboxylase is much higher (40- to 50-fold) than that in C, plants, consistent with its key role in net CO* fixation by the Cq pathway of photosynthesis (5-7). The high activity of this bundle sheath chloroplast enzyme is in contrast to that of mitochondrial NAD-malic enzyme, which is dramatically modulated by acetyl-CoA and FruP, (8). Many of the previous investigations dealing with the regulatory properties of NADP-malic enzyme have been focused on the inhibitory effects of various metabo1 This is paper No. 49 in the series “Structure and lites on enzyme activity (9-12). However, Function of Chloroplast Proteins” and No. 48 is Ref. (1) by Asami et al. The research was supported in part it should be noted that the concentrations by the grants from the Ministry of Education of Japan of the compounds exhibiting inhibitory ef(No. 310413), the Toray Science Foundation (Tokyo), fects were much higher than their physiological levels. On the other hand, Johnson and the Nissan Science Foundation (Tokyo). * Recipient of the Postdoctoral Fellowship from the and Hatch (13), using a partially purified Japan Society for the Promotion of Science (JSPS) preparation of maize leaf NADP-malic 1978. enzyme, demonstrated that enzyme activity s Abbreviations used: DTE, dithioerythritol; FruP,, fluctuates quite appreciably in response to a fructose 1,6-bisphosphate; a-KG, a-ketoglutarate; change in pH of the reaction mixture (see OAA, oxaloacetate; PEP, P-enolpyruvate; 3-PGA, 3also Fig. 6 of (1)). phosphoglycerate; RuP,, ribulose l&bisphosphate; In the work reported in this communicaSUP,, sedoheptulose 1,7-bisphosphate; Tricine, Ntion, we have examined the interaction beTris(hydroxymethyl)methyglycine. 581

0003-9861/79/100581-07$02.00/O Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.

582

ASAMI, INOUE, AND AKAZAWA

tween pH, Mg2+, and malate in modulating the activity of NADP-malic enzyme purified from maize leaves (1). The results are discussed with respect to the possible regulatory properties of the enzyme during Cq photosynthesis. MATERIALS

AND METHODS

Materials. NADP, oxaloacetate, sugar phosphate, and DTE were purchased from Boehringer-Mannheim; Tricine and NADPH from Sigma; Na-Lmalate from Wako Chemical Company Ltd. (Tokyo); and pyridoxal5’-phosphate and thiamine pyrophosphate from Calbiothem. All other chemicals used were of reagent grade. Enzyme assay. NADP-malic enzyme was isolated and purified from 3- to I-week-old maize leaves as described previously (1). Unless noted otherwise, the standard assay mixture contained the following components in millimolar concentrations: Tricine-NaOH (pH 7.5, 7.9, 8.4), 50; DTE, 5; Lmalate and MgCl,, as specified; NADP, 0.4; and enzyme (0.1-0.2 unit) in a total volume of 0.5 ml. The reaction was carried out at 25°C and the increase in absorbance at 340 nm due to the formation of NADPH in the forward reaction (decarboxylation) was recorded using a Gilford Model 250 spectrophotometer (1). The specific activity of the homogeneousenzyme was 100pm01 malate decarboxylatedimg protein. min. RESULTS AND DISCUSSION

PH, M#+, and Malate Studies in several laboratories have elucidated that different regulatory factors modulate activities of the enzyme reactions in the Calvin-Benson cycle: (a) change in the stromal environment during dark/light transients (14- 16), (b) activation by the ferredoxin/thioredoxin system (17, 18) and/or “light effect mediator” (19), and (c) effect of metabolites such as NADPH and ATP (20, 21). As to case (a), it was reported that upon illumination of dark-treated intact C, chloroplasts an increase in photosynthetic CO,-fixing activity occurs which is ascribed to light-induced changes in two environmental factors, pH and Mg2+; during dark/ light transients stromal pH increases from 7.2 to 8.1 and levels of free Mgz+ increase by about 3-5 mM (14, 15). Light activation of certain Calvin-Benson cycle enzymes, e-g., FruP,-ase, RuP, carboxylase, and SUP,-ase, appears to be due, at least in part, to these light-induced changes in stromal

pH and Mg2+ (16). Although there are no data available concerning dark/light transients in pH and Mg2+ in the bundle sheath chloroplasts of Cq plants, it is not unreasonable to postulate that the same mechanism(s) of light activation is applicable to the bundle sheath Calvin-Benson cycle. One can thus envision that changes in pH and Mg2+ might be of importance in regulating NADP-malic enzyme which supplies COZ to bundle sheath RuP, carboxylase. Furthermore it is possible that the concentration of malate in the bundle sheath chloroplast modulates the activity of NADPmalic enzyme, although once again no precise quantitative data are available concerning the stromal malate concentration. Hatch (22) estimated the malate concentration to be about 2.4 mM in whole bundle sheath cells of maize. Bearing these previous findings in mind, we first examined the activity of NADPmalic enzyme as a function of malate and Mgz+ concentration in the pH range of 7.5 to 8.4 at a constant level of NADP (0.4 InM). At pH 8.4 in the presence of 5 mM Mg2+, the K, (malate) was 182 PM, whereas at 1 mM Mg*+ malate exhibited a slight inhibitory effect at the highest concentration examined, with a K, (malate) of 235 FM (Fig. 1A). At pH 7.9, substrate inhibition by malate occurred at both 1 and 5 mM Mg2+, the K, (malate) values being 185 and 110 PM, respectively (Fig. 1B). At pH 7.5 and malate levels greater than 1 mM a prominent substrate inhibition was observed in the presence of l-10 mM Mg2+. At 1 mM Mg2+, NADP-malic enzyme activity was maximal at 0.4 mM malate and at 0.8 mM in the presence of 5 or 10 mM Mg2+, with the K, (malate) being 110 PM (1 lllM Mg2+) and 78 PM (5 or 10 mM Mg2+) (Fig. 1C). These results, in essence extending the previous finding by Johnson and Hatch (13), indicate that both the apparent activity of the enzyme for malate and the degree of substrate inhibition increase with decreasing pH and low Mgz+ concentration. We next studied NADP-malic enzyme activity as a function of Mg2+ concentration at a fixed level of malate (3 mM) and NADP (0.4 InM), and the results are shown in Fig. 2. It is apparent that under the above assay

2

3

I

6Malate

I

a

2 ( mM 1

3

r++-

6

13

I

2

3

6

13

FIG. 1. Effect of malate concentration on NADP-malic enzyme activity as a function of pH and Mg *+. The reaction was started by adding 0.4 mM NADP to the standard assay mixture adjusted to pH 8.4,7.9, or 7.5, and followed spectrophotometrically at 25°C. Other experimental details are described under Materials and Methods.

I

584

ASAMI, INOUE, AND AKAZAWA

O.‘l-----l 0.12-

/I

4 l/Mg”+

8

pH 7.5

12

J

16

(I/mM)

FIG. 2. Double-reciprocal plot of the relationship between NADP-malic enzyme activity and MgZ+ concentration at pH 8.4 and pH 7.5. The reaction was started by adding 3 mM malate to the standard assay mixture adjusted to pH 7.5 or 8.4. Other experimental details are described under Materials and Methods.

conditions the V,,, at pH 7.5 is about onehalf that at pH 8.4 and that there is marked substrate inhibition at pH 7.5 (Fig. 1A vs 10 K, (Mg2+> was determined to be 63 PM at pH 8.4 and 210 PM at pH 7.5, indicating a marked increase in the apparent affinity of the enzyme for Mg2+ at higher pH. From these results it is hypothesized that the activity of NADP-malic enzyme, the major decarboxylase which supplies CO, to bundle sheath RuP, carboxylase in NADP-malic enzyme-type Cq plant, is controlled by multiple factors, including PH, Mg’+, and malate. Thus we propose that under the conditions in which enzymes of the Calvin-Benson cycle are light activated (i.e., high stromal pH and Mg2+), substrate (malate) inhibition of NADPmalic enzyme is reduced, and this decrease is associated with a concomitant increase in enzyme activity which is attributed to increases in pH and Mg2+ concentration.

With respect to the cooperative interaction between the bundle sheath cells and the surrounding mesophyll cells during C, photosynthesis, conditions in which photosynthetic CO, fixation in the bundle sheath is prevented, substrate inhibition of NADPmalic enzyme may cause a decrease in the rate of malate decarboxylation, resulting in a decrease in the level of pyruvate, which is required for PEP regeneration and thus CO, fixation in the mesophyll. In addition, an increase in malate concentration could lead to feedback inhibition of PEP carboxylase (23). Thus, in addition to the light activation of the key Cq cycle mesophyll enzymes, pyruvate, Pi dikinase (24), and NADP-malate dehydrogenase (13), the experimental findings described above suggest that the bundle sheath enzyme, NADPmalic enzyme, indirectly regulates photosynthetic carbon metabolism in the mesophyll. NADPHINADP

Ratio

It is well documented that the agranal bundle sheath chloroplasts of NADP-malic enzyme-type Cq plants are deficient in photosystem II activity and thus also in their ability to photoreduce NADP from water (25, 26). It is frequently suggested that NADP-malic enzyme provides both CO, and reducing power (NADPH) for the operation of the Calvin-Benson cycle in the bundle sheath (2’7, 28). It is shown that malate decarboxylation is coupled to the reduction of 3-PGA in isolated maize bundle sheath strands (27-29). Therefore, in an attempt to further elucidate the possible relationship between the NADP-malic enzyme reaction and the production of NADPH we determined the activity of the enzyme as a function of the NADPH/NADP ratio from zero (the standard condition) to 2.0 (Fig. 3). It should be pointed out that in this reaction system, concentration of NADPH plus NADP is fixed at 0.2 InM, yet maintaining the concentration of NADP required for the malate decarboxylation at the saturating level (cf. (1)). The results indicate that the higher the ratio of NADPH/NADP, the lower the enzyme activity, and this inhibitory effect increases with decreasing

REGULATORY

PROPERTIES

OF MAIZE

NADP-MALIC

ENZYME

585

deficient in photosystem II. However, the finding that the activity of NADP-malic enzyme is less sensitive to higher NADPH/ NADP ratio at pH 8.4 than at pH 7.5 suggests that the enzyme may also be regulated by an interaction between stromal pH and the levels of NADPH and NADP. Effect of Metabolites

The effects of various metabolites on the activity of NADP-malic enzyme were tested under two assay conditions: (A) 5 mM o1.5 2.0 1.0 0.5 0 malate, 5 mM Mg’+, and 0.4 mM NADP at NADPH / NADP Ratio pH 8.4; and (B) 1 mM malate, 5 mM Mg’+, FIG. 3. Effect of the NADPH/NADP ratio on NADPand 0.4 mM NADP at pH 7.5. The following malic enzyme activity. The reaction was started by compounds at 2 mM did not exhibit any efadding 3 mM malate to the assay mixture. The con- fect on enzyme activity within a range of centrations of NADPH plus NADP and Mgz+ were +lO% in both assay system; aspartate, fixed at 0.2 and 5 mM, respectively. Enzyme acglutamate, 3-PGA, glyceraldehyde 3-P, tivity (PmoYmg protein. min) at NADPH/NADP ratio dihydroxyacetone-P, ribose 5-P, FruP,, of zero was 69.3 at pH 8.4, 69.2 at pH 7.9, and 47.6 at citrate, and isocitrate. In system (A) neither pH 7.5. ATP, ADP, PEP, glycolate, glutamine, serine, glycine, xylulose 5-P, RuP,, thiapH. It has been reported by Lendzian and mine pyrophosphate, nor 0.3 mM pyridoxalBassham (30) that in illuminated spinach 5’-phosphate inhibit enzyme activity. On chloroplasts the ratio of NADPH/NADP is the other hand, the following compounds approximately 1.8; however, such a meas- showed an inhibitory effect in system (B) at urement has not been performed with maize 2 mM: ATP (40%), ADP (28%), PEP (33%), leaf bundle sheath chloroplasts which are and glycolate (17%).

_ B.p+lr.s pyr”“at.

0

Effector

( mM 1

I

Effector

2

3

4

(mM)

FIG. 4. Effect of ol-keto acids on NADP-malic enzyme activity at (A) pH 8.4 and(B) 7.5. The reaction was started by adding (A) 1 mrd or (B) 5 mM malate to the assay mixture at 25°C. The concentration of Mg2+ and NADP was fixed at 5 and 0.4 mM, respectively. Control enzyme activity was (A) 83.2 and (B) -,T,, 1, ou.u pmoumg protem mm.

586

ASAMI, INOUE, AND AKAZAWA

The results given in Fig. 4 show the inhibitory effect of various a-keto acids on the NADP-malic enzyme activity. The inhibition by glyoxylate, OAA, and a-KG is more pronounced at pH 7.5 than at pH 8.4. However, no end-product-type inhibition was observed following the addition of pyruvate. The similar result has been reported previously using a partially purified malic enzyme from maize leaf (10). A doublereciprocal plot of the data (Fig. 5) indicates a mixed-type inhibition by OAA and an uncompetitive type inhibition by a-KG with respect to malate at both pH 7.5 and 8.4. At pH 7.5 4 lllM HCO,- did not exhibit any inhibitory effect. Using the bundle sheath chloroplasts isolated from NADP-malic enzyme type plants, Rathnam and Edwards (28) reported that HCO,- exhibits an inhibitory effect on the light-dependent and 3PGA-stimulated malate decarboxylation reaction. It has been estimated by Hatch and Osmond (4) that the concentration of “CO2 + HC03-” in the bundle sheath cells is approximately 0.6 mM during active Cqphotosynthesis. There are some discrepancies between our present experimental results and those reported by Bhagwat and Sane (11, 12), however. For instance, as opposed to the latter authors we could not find any allo-

steric effect of FruP,, and OAA was found to exhibit a mixed-type inhibitory effect instead of competitive-type inhibition. Reasons for such disagreement are not immediately clear, but different assaying conditions, e.g., malate levels, might have probably caused discrepancies. Concluding

Remarks

Both the content (approx. 1.2% of the total soluble protein) and specific activity of NADP-malic enzyme in maize leaf tissue are quite high (1); therefore it is likely that the regulatory properties of this bundle sheath chloroplast enzyme are different from those of other enzymes in the Calvin-Benson cycle (14-19). NADP-malic enzyme activity appears to be modulated by an interaction between pH, Mg2+, and malate; at pH 7.5 the enzyme is subject to substrate (malate) inhibition as well as an enhanced sensitivity to the high NADPH/NADP ratio. Conversely, under conditions of pH and Mg2+ similar to those presumably occurring in an illuminated chloroplasts NADP-malic enzyme would attain its maximal activity at high levels of malate. However, it should be noted that the proposed regulatory properties of this important bundle sheath enzyme are based largely on the assumption that the

/P

I

o.,o-

0.05

L

I 2

4 I / Malote

6 (I/mM)

I3

1 IO

-

10 I / Malote

( I /mM

)

FIG. 5. Double-reciprocal plot of the relationship between NADP-malic enzyme activity and malate concentration at (A) pH 8.4 and (B) pH 7.5 in the absence (control) and presence of OAA, a-KG, or HC03-. The reaction was started by adding 0.4 mM NADP to the standard assay mixture containing 5 rnM MgZ+.

REGULATORY

PROPERTIES

OF MAIZE

pH and levels of Mg2+ and malate in the bundle sheath chloroplasts of maize are perturbed during dark/light transients. This assumption remains to be verified by direct experimentation with isolated maize bundle sheath cells and chloroplasts. ACKNOWLEDGMENT

The authors wish to record their sincere thanks to Dr. Ray Chollet for critical reading of the manuscript and invaluable discussions in connection with this investigation. REFERENCES 1. ASAMI, S., INOUE, K., MATSUM~TO, K., MURACHI, A., AND AKAZAWA, T. (19’79)Arch. Biochem. Biophys. 194, 503-510. 2. BLACK, C. C. (1973) Annu. Rev. Plant Physiol. 24, 253-286. 3. HATCH, M. D. (1976) in CO* Metabolism and

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