Interaction Between Chloroplast Phosphoglycerate Kinaseand Glyceraldehyde-3-Phosphate Dehydrogenase

INTERACTION BETWEEN CHLOROPLAST PHOSPHOGLYCERATE KINASE A N D G LYC E RA L DE HY DE-3- PH0sPHATE DEHYDROGENASE

Louise E. Anderson, Xiao-yi Tang, Cote Johansson, Xingwu Wang, lvano A. Marques, and Jerzy Macioszek

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 INTRODUCTION.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 PHYSIOLOGICAL INDICATIONS OF INTERACTION . . . . . . . . . . . 274 KINETIC INDICATIONS OF INTERACTION . . . . . . . . . . . . . . . . 275 PHYSICAL EVIDENCE FOR INTERACTION . . . . . . . . . . . . . . . . 275 EVIDENCE FOR INTERACTION BETWEEN THESE TWO ENZYMES IN OTHER SYSTEMS . . . . . . . . . . . . . . . . . . . . . . 278 VI. CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 I. 11. 111. IV. V.

Advances in Molecular and Cell Biology Volume 15A, pages 273-279. Copyright 0 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0 114-7

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ANDERSON, TANG, JOHANSSON, WANG, MARQUES, and MACIOSZEK

ABSTRACT Physiological, kinetic and physicochemical evidence suggests interaction between the Calvin cycle enzymes P-glyceratekinase and glyceraldehyde-3-Pdehydrogenase.

1. INTRODUCTION P-glycerate kinase and glyceraldehyde-3-P dehydrogenase together accomplish the most important reaction of the reductive pentose phosphate (Calvin) cycle, the reduction of the carboxylic acid P-glycerate to the phosphate sugar, glyceraldehyde3-P. The intermediate in the two enzyme reaction, 1,3-P2-glycerate, is unstable and, in fact, has not been detected among the products of CO, fixation. Here we review indications for interaction between pea leaf chloroplast P-glycerate kinase and glyceraldehyde-3-P dehydrogenase and present new evidence for the physical interaction between these two enzymes.

II. PHYSIOLOGICAL INDICATIONS OF INTERACTION The potential activity of 6, of the 11, Calvin cycle enzymes, calculated by assuming free diffusion of substrate between enzymes, is too low to account for observed levels of CO, fixation in pea chloroplasts (Marques et al., 1987). Glyceraldehyde3-P dehydrogenase is apparently twice as active as would be predicted if P,-glycerate diffuses to it through the stroma from P-glycerate kinase (Table I ) . If however the two enzymes do interact, then the activity of the two enzymes should be more than sufficient to support photosynthetic CO, fixation. These physiological experiments, then, suggest that these two enzymes might be interacting in the chloroplast stroma. Table 1. Maximal and Estimated In Situ Activities of Strornal Enzymes Involved in

Glyceraldehyde-3-P Metabolism in Pea Chloroplasts Activiry Observed

COz Fixation P-Glycerate Kinase Glyceraldehyde-3-P Dehydrogenase Triose-P lsomerase Aldolase Transketolase Note

0.03 1.05 0.58 0.1 I 0.28 0.26

Potenrial Aclivit>Jiri Stroma

0.14 0.03 0.082 0.0069 0.0013

Activih Fraction qf Required,for. Required PGA EXPO,? Activity

0.05 0.05 0.02 0.0 1 0.01

2.8 0.6 4.1

0.69 0.13

From Marques et al. (1987). with permission. Potential activity is the estimated activity at substrate concentrations measured in these experiments. For details see Marques et al. (19x7).

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111. KINETIC INDICATIONS OF INTERACTION In experiments with crude stromal extracts we found what appeared to be negative cooperativity when the concentration of P-glycerate was varied and the activity of the dehydrogenase assayed (Macioszek and Anderson, 1987). The negative cooperativity can be explained in several ways, one possibility being that there are two different forms of glyceraldehyde-3-P dehydrogenase present in the cuvette. These different forms of the enzyme could include the enzyme complexed with P-glycerate kinase. In experiments with the purified enzymes we again found negative cooperativity when P-glycerate concentrations were varied (Macioszek et al., 1990). Here we found that if we held the concentration of glyceraldehyde-3-P dehydrogenase constant and varied P-glycerate kinase in the presence of high levels of the Calvin cycle substrates ATP and P-glycerate (and reduced pyridine nucleotide) the kinetics were Michaelis-Menten, as if P-glycerate kinase (or P-glycerate kinase complexed with product P2-glycerate)was acting as substrate for glyceraldehyde-3-P dehydrogenase. Kmp-g,ycerate kinase values obtained for chloroplastic glyceraldehyde-3-P dehydrogenase were 34 ? 2 pM when NADPH was the pyridine nucleotide substrate and 54 k 7 pM when NADH was the pyridine nucleotide substrate. Under these conditions, it appears that transient complex formation between these two enzymes might occur. For other possible explanations of these data see Macioszek et al. ( 1 990).

IV. PHYSICAL EVIDENCE FOR INTERACTION If the kinetic experiments are indicative of interaction then one might also be able to detect physical interaction between these two enzymes. Fluorescence polarization has been used extensively to study protein interaction including the interaction between several glycolytic enzymes. Association results in a larger fluorescent species and hence a decrease in depolarization when the fluorophore is excited by polarized light. We therefore looked for an increase in polarization and anisotropy in experiments in which glyceraldehyde-3-P was labeled with fluorescein isothiocyanate (FITC). The results of these experiments are shown in Figure 1. Clearly, addition of P-glycerate kinase causes an increase in anisotropy, indicating that the two proteins are forming a complex. These results together with the kinetic experiments indicate that these two enzymes interact both in the presence and absence of substrate. Notably, the two enzymes are found at the same active site concentration in the intact chloroplast (about 30 pM, or 30 pM P-glycerate kinase, 7.5 pM glyceraldehyde-3-P dehydrogenase tetranier). A 1 : 1 complex is therefore possible. In the fluorescence anisotropy experiments the concentration of the enzymes was 3 orders of magnitude higher than in the experiment in which the P-glycerate kinase concentration was varied and glyceraldehyde-3-P dehydrogenase activity was followed. It seems possible that there is interaction between these enzymes both in

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0.07

1

0.061

0.05

I

0

I

0.1 0.2 0.3 0.4 0.5 0.6 Phosphoglycerate Klnase (wM)

Figure 1. Effect of chloroplastic P-glyceric kinase on fluorescence anisotropy of fluorescein isothiocyanate (FITC) labeled pea leaf glyceraldehyde-3-P dehydrogenase. NADP-linked glyceraldehyde-3-P dehydrogenase isolated from pea leaves by the method of Li and Anderson (1992) was labeled with (FITC) a5 described by Ovadi et al. (1 983). Excitation was at 495 nm and emission was monitored at 51 8 nrn (Hitachi F-4010 Spectrofluorimeter equipped with polarizer). The cuvette contained potassium phosphate buffer (10 mM, pH 7.01, EDTA (0.5 mM), mercaptoethanol (3.5 mM) and glyceraldehyde-3-P dehydrogenase (0.9 pM, 3.7 pM active site, 2.6 mol FITC per glyceraldehyde-3-P dehydrogenase tetramer) and P-glycerate kinase at the concentrations indicated. The P-glycerate kinase used in these experiments flunrecced when excited with 495 nm light, presumably due to bound tannins. At the highest levels of P-glycerate kinase used in these experiments the contribution of the kinase to the total fluorescence was calculated to be less than 1YO and the contribution to the anisotropy less than 5Y0, without interaction. This experiment was repeated with a second batch of FITC-labeled glyceraldehyde-3-P dehydrogenase and similar results were obtained.

the absence and in the presence of their common substrate, but that the interaction between the enzymes is greater in the presence of 1,3-P2-glycerate. In the experiments described in Figure 2 we found both an increase in phosphoglycerate kinase activity and a shift in the partition coefficient when glyceraldehyde-3-P dehydrogenase was partitioned together with the kinase in a polyethyleneglycol-dextran countercurrent phase partitioning system. In this system the partition coefficient for glyceraldehyde-3-P dehydrogenase was 0.08f0.02 (mean of 6 determinations) and, for P-glycerate kinase, 0.7 (6 determinations). The partition coefficient for the complex, 0.03 f 0.01 (mean of 5 determinations) was close to the product of the partition coefficients for the individual enzymes, and

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I

0

2

4

6

8 10 12 Tube Number

14

16

18

Figure 2. Countercurrent distribution of P-glycerate kinase and glyceraldehyde-3-P dehydrogenase from pea chloroplasts. P-glycerate kinase (m) partitioned in the presence of glyceraldehyde-3-P dehydrogenase and P-glycerate kinase partitioned alone (0). Glyceraldehyde-3-P dehydrogenase ( 0 )partitioned alone. Partitioning was carried out essentially as described by Skrukrud et al. (1991) at roo! temperature in a 60 chamber centrifugal counter-current distribution apparatus (Akerlund, 1984). The aqueous two-phase system contained 5% (wt/wt) dextran T-500, 8.6% (wt/wt) PEG8000, 50 m M potassium phosphate (pH 7.8), 5 m M DTT. This gave a top to bottom phase ratio of 3.1 :l. The capacity of each bottom chamber was 0.72 ml. Each chamber was loaded with 2.95 ml of the phase system. Enzyme(s)was added to the first of 20 chambers. Nineteen transfers were performed. Between each transfer there was a shaking time of 2 min followed by a centrifuging period of 4 min. At the beginning of the experiment chamber 0 contained 5.8 pg glyceraldehyde-3-P dehydrogenase, or 160 ng P-glycerate kinase, or both, for a molar ratio of 10 dehydrogenase:l P-glycerate kinase and an active site ratio of 40:l. We saw marked shifting of P-glycerate kinase in the presence of glyceraldehyde-3-P dehydrogenase in 10 of 13 experiments. In 7 (of the 10) we also saw an increase (average 5-fold) in kinase activity in the first fractions (i.e. in the dextran phase with the dehydrogenase). Changes in the partitioning of glyceraldehyde-3-P dehydrogenase, if any, were not detectable.

therefore (see Albertsson, 1986) there was probably a 1: 1 association of the two enzymes, little masking of charged groups when the enzymes were associated, and the contact area was not large. We saw no effect of glyceraldehyde-3-P dehydrogenase on the partitioning of triose-P isomerase with concentrations of glyceraldehyde-3-P dehydrogenase 200-fold higher than those used in this experiment (data not shown). The effect of chloroplastic glyceraldehyde-3-P dehydrogenase on the partitioning of P-glycerate kinase is then not a simple protein effect: There appears to be specificity in the interaction.

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V. EVIDENCE FOR INTERACTION BETWEEN THESE TWO ENZYMES IN OTHER SYSTEMS There is evidence for complex formation between these two enzymes from other plant and animal systems. Gontero et al. (1988) have reported the isolation of a multienzyme complex of several Calvin cycle enzymes including P-glycerate kinase and glyceraldehyde-3-P dehydrogenase from spinach. This complex has not been fully characterized and might in fact represent several different complexes rather than one large complex. In gel filtration experiments, Malhotra et al. (1987) found evidence for complexing between cytosolic P-glycerate kinase and glyceraldehyde-3-P dehydrogenase isolated from mung beans. Immunocytochemical experiments indicate that P-glycerate kinase and glyceraldehyde-3-P dehydrogenase are localized in the same region in the prokaryote Zvmonzoizns rizobilis and may therefore be complexed or be part of a larger glycolytic complex (Aldrich et al., 1992). Extensive kinetic studies carried out with the enzymes isolated from halibut muscle suggest that kinase-bound P,-glycerate acts as substrate for the dehydrogenase (Weber and Bernhard, 1982). Species specificity in the association of P-glycerate kinase with immobilized glyceraldehyde-3-P dehydrogenase and a species-dependent increase in the steady-state rate of the coupled two-enzyme reaction has been reported by Khoroshilova et al. (1992). Our data are consistent with all of these experiments and suggest that P-glycerate kinase and glyceraldehyde-3-P dehydrogenase interact both in glycolysis and gluconeogenesis.

VI. CONCLUSION We have physiological, kinetic, and physical evidence for interaction between the Calvin cycle enzymes P-glycerate kinase and glyceraldehyde-3-P dehydrogenase. The advantage to be gained by this interaction would include the stabilization of the labile intermediate P,-glycerate and enhancement of the overall rate of CO, fixation, since glyceraldehyde-3-P dehydrogenase is not rate limiting if it is complexed with, or accepts substrate from P-glycerate kinase, and a reduction in the lag time in photosynthetic induction, which is thought to represent the time required for the buildup of photosynthetic intermediates, including P,-glycerate, to steady state levels.

ACKNOWLEDGEMENTS Support for this work came from the University of Illinois-ChicagoResearch Board, the US Department of Energy (Contract DE-AC02-78EV04961), the US National Science Foundation (Grants PCM 84-17081, DCB 90-18265 and INT 91-15490), and the Chinese National Science Foundation (Grant 39230050).We are indebted to Da Xua Ming (Shanghai Institute of Plant Physiology, Chinese Academy of Science), Luo Fang and Zhao Qi (Shanghai Research Centre of Biotechnology, Chinese Academy of Science) for excellent

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technical assistance, and to Li Chang-hou (Shanghai Research Centre of Biotechnology) for the use of the fluorimeter.

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