6-Phosphofructo-2-kinase and D -fructose-2,6-bisphosphatase activities in mung bean seedlings

Biochimica etBiophysicaActa, 798 (1984) 317-324

317

Elsevier BBA21728

6-PHOSPHOFRUCTO-2-KINASE AND D-FRUCTOSE-2,6-BISPHOSPHATASE ACTIVITIES IN MUNG BEAN SEEDLINGS GAD AVIGAD and PRISCILLA J. BOHRER

Department of Biochemistry, UMDNJ- Rutgers Medical School, Piscataway, NJ 08854 (U.S.A.) (Received August 4th, 1983)

Key words: Fructose2,6-bisphosphate; Fructose-6-phosphate- 2.kinase; Fructose-2,6-bisphosphatase,. (Mung bean)

6-Phosphofructo-2-kinase (ATP: D-fructose-6-phosphate-2-phosphotransferase) and D-fructose-2,6-bisphosphatase activities have been found in extracts prepared from etiolated mung bean seedlings. The activity of 6-phosphofructo-2-kinase exhibits a sigmoidai shape in response to changes in concentrations of both substrates, D-fructose 6-phosphate and ATP (So.5 values of 1.8 and 1.2 mM, respectively). Inorganic orthophosphate (Pi) has a strong stimulating effect on the 2-kinase activity (A o.5 at about 2 raM), moderately increasing the Vm,x and modifying the response into hyperbolic curves with K m values of 0.4 and 0.2 mM for fructose 6-phosphate and ATP, respectively. 3-Phosphoglycerate (I0. s about 0.15 mM) partially inhibited the kinase activity by counteracting the Pi activation. In contrast, the activity of D-fructose-2,6-hisphosphatase (K m 0~38 mM) is strongly inhibited by Pi (10.5 0.8 mM) lowering its affinity to fructose-2,6-P2 (K m 1.4 raM). 3-Phosphoglycerate activates the enzyme (A0. 5 at about 0.3 mM) without causing a significant change in its K m for fructose-2,6-P2. The activities of both of these enzymes in relationship to the metabolic role of D-fructose 2,6-bisphosphate in the germinating seed is discussed.

Introduction t-o-fructose 2,6-bisphosphate has been recently described as a key metabolite that plays a critical role in the regulation of carbohydrate metabolism (for reviews see Refs. 1-3). Whereas most of the detailed studies about the biochemistry of fructose-2,6-P 2 have been carried out with mammalian tissues, predominantly with the hepatocyte, several observations also suggest involvement of this sugar in the control of organic carbon fluxes in plants. Sabularse and Anderson [4,5] were first to detect fructose-2,6-P2 in plant tissues as well as its ability to stimulate PPi:fructose-6-phosphate-l-phosphotransferase activity (EC 2.7.1.90) [4,5]. This activation effect was subsequently confirmed in several other studies [6-9]. Fructose-2,6-P2 also inhibited the activity of plants D-fructose-l,6-bisphosphatase (EC 3.1.3.11) [6,10,11] and stimu0304-4165/84/$04.00 © 1984 Elsevier Science Publishers B.V.

lated the potato tubers UDP glucose phosphorylase [12]. An activation effect by fructose-2,6-P2 on the plastid 6-phosphofructose-l-kinase (ATP:Dfructose-6-phosphate-l-phosphotransferase, EC 2.7.1.11) from Ricinus endosperm [12] could not be established for the spinach-leaf enzyme [6]. The intracellular concentration of fructose-2,6P2 in green leaves was estimated to be about 5 #M, or even higher (up to 30 nmol/g fresh tissue) and the sugar is assumed to reside in the cytosol [6,10]. Similar values (20-60 nmol/g fresh tissue) were also found in etiolated legume seedlings (Avigad, G., unpublished data). This concentration is of a similar level at which other sugar phosphate intermediates are present in the plant tissue and obviously indicates the presence of effective specific enzymes responsible for the synthesis as well as hydrolysis of fructose-2,6-P2. It is of interest to establish whether this enzymic entity in plants is

318 comparable to the specific 6-phosphofructose-2kinase/fructose-2,6-bisphosphatase complex identified in the hepatocyte [1-3, 14-18]. The present study describes several experiments which establish the presence of these enzymic activities in extracts prepared from mung bean (Phaseolus aureus) seedlings. Some kinetic properties of these enzymic reactions and their modulation by important metabolic intermediates have been explored. A report demonstrating the presence of 6-phosphofructo-2-kinase in spinach leaves appeared while this manuscript was in preparation [19]. Our study with the etiolated seedlings generally confirm and extend the observations presented in the study of the photosynthetic tissue. Materials and Methods Purified enzymes and biochemical reagents used were purchased from Sigma Biochemical Co. Fructose-2,6-P2 was synthesized chemically [20] using ammonium borate as the eluant in the last chromatographic separation step [11]. In later stages of the experimental work, samples of fructose-2,6-P2 were purchased from Sigma. Partially purified PPi:fructose 6-phosphate-l-phosphotransferase (a DEAE-cellulose fraction, spec. act. of 0.3 units/mg protein) was prepared from mung bean seedlings [5]. 5-day-old etiolated mung bean sprouts were purchased in a local produce market. Spectrophotometric and colorimetric measurements were taken in a Gilford model 240 recording spectrophotometer using quartz cuvettes with a 1.0-cm light path. The colorimetric resorcinol reaction for fructose [21] was modified by reducing the volume of the reaction system, by increasing its sensitivity to allow the measurement of smaller amounts of ketoses or ketosyls and by adjusting the procedure for the determination of the sugar also in the presence of borate. This colorimetric method was also used to verify the concentration of stock solutions of the various fructose derivatives used as substrates in this study. The stock resorcinol reagent was composed of (in mg/ml): resorcinol, 1.5; thiourea, 2.0; sodium tetraborate, 2.0; and sulfamic acid, 10.0, all dissolved in glacial acetic acid. The solution had a useful shelf-life of at least 3 months when kept in a

brown bottle at room temperature. A freshly made dilution made by mixing 2 volumes of the stock resorcinol solution with 13 volumes of analytical grade concentrated HCI was prepared daily to provide the reagent used for the colorimetric assay. The determination of fructose (as well as fructosyls or fructose phosphates) is carried out as follows: Samples of ketose, 5-200 nmol, in a total volume of 0.4 ml are placed in 10 × 75 mm glass test-tubes. Diluted resorcinol reagent (0.8 ml) is added to each tube and the solutions mixed. The test tubes are heated at 80°C for 10 min in a heating block (preset at 82°C) and then immediately cooled down in ice-cold water. Absorbance at 515 nm is read against a reagent blank. A linear correlation between absorbance and the amount of fructose present in the range specified above, is observed, with a specific molar absorption value (%1s) of 4500.

Assay of 6-phosphofructo-2-kinase The production of fructose-2,6-P2 was measured in earlier experiments by the activation patterns of rabbit muscle 6-phosphofructo-l-kinase [22,23] but later by using the highly sensitive activation effects on the mung bean seedlings PP~:D-fructose-6-phosphate-l-phosphotransferase reaction [5-7]. However, for the purpose of assaying a large number of samples generated by carrying out kinetic experiments, the colorimetric resorcinol procedure, even though less sensitive than the enzyme activation assay, was often found to be useful and having a corresponding accuracy for analyzing fructose-2,6P2 in most of the ranges of substrate concentrations employed in the present system. The basic reaction mixture (1.0 ml) contained (in /zmol): 4-(2-hydroxyethyl)-l-piperazineethansulphonic acid (Hepes) buffer pH 7.4, 100; fructose-6-P, 1.0, ATP. 2; MgC12, 3; NaF, 10; dithiothreitol, 0.5; EDTA, 0.5; and a sample of enzyme solution to be assayed. Incubated at 30°C, aliquots (usually 200 /xl) are withdrawn at various time intervals into a set of test tubes and 20/~1 of freshly prepared 100 m g / m l NaBH 4 solution is added to each sample. This treatment completely removed all ketoses such as fructose-6-P and fructose-l,6-P2 which could interfere with the colorimetric assay of fructose2,6-P2. After 20 to 30 min, 180 #1 of glacial acetic

319 acid are added. Within several minutes, when the foaming produced as the result of decomposition of borohydride subsided, 0.8 ml resorcinol reagent is added and the samples analyzed for ketose content as described above. If required, reduced samples can be stored in the cold or frozen prior to carrying out the colorimetric assay at a later date. Readings at 515 nm are made against reagent blanks lacking enzyme, to verify the complete reduction of the initial fructose-6-P present and to record the base value of fructose content which may still be present in the partially purified enzyme preparation. This endogenous ketose may be a contribution of a fructan or of a trace of sucrose not removed completely by dialysis. 6-Phosphofructo-l-kinase was assayed in a system (1.0 ml) containing (in ~tmol): Hepes buffer, pH 7.4, 100; dithiothreitol, 2; EDTA, 0.4; ATP 2; MgC12, 2; fructose-6-P, 0.5; NADH, 0.2, muscle aldolase (0.25 units), glycerophosphate dehydrogenase (0.5 units), and triose phosphate isomerase (2 units). Reaction is started by addition of a sample of the kinase to be assayed, and following it spectrophotometrically at 340 nm for about 30 min. PPI: fructose-6-phosphate-l-phosphotransferase was assayed in a system similar to that of the 6-phosphofructo-l-kinase but in which ATP was substituted by 2 #mol sodium pyrophosphate, fructose-6-P was present at 1.0 mM, and 50 nmol fructose-2,6-P2 added for activation [5-7]. DFructose-2,6-bisphosphatase was assayed in a system (1.0 ml) containing (in nmol): Tris-C1 pH 8.2, 40; fructose-2,6-P2, 0.5; dithiothreitol, 0.4; MgC12, 1.0; EDTA, 0.4; NADP ÷, 2; phosphoglucoisomerase (0.55 units), yeast glucose 6-phosphate dehydrogenase, (0.55 units) and a sample of the bisphosphatase to be assayed. Reaction is initiated by addition of fructose-2,6-P2 followed up at 340 nm for about 30 rain. Control experiments have indicated that under these assay conditions contribution of 6-phosphogluconate dehydrogenase activity in the extracts could not account for more than 10% of the rate of NADP + reduction reported. Subsequent experiments coupled with equivalent amount of N A D + and Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase, resulted in values similar to those observed with the NADP+-dependent yeast enzyme. D-Fructose1,6-bisphosphatase was assayed as described above,

but replacing fructose-2,6-P2 by 1 mM fructose1,6-P2 as the substrate. A unit of enzyme activity in all assays is equivalent to a conversion of 1 ~mol substrate per min at the conditions specified. Protein was determined colorimetrically [24] using bovine serum albumin as the standard. Preparation of extracts

Washed seedlings, usually in batches of 100 g, were homogenized at 4°C for 1 min in an Oster high speed blendor in 100 ml 0.1 M Tris-Cl buffer, pH 8.0 containing 2 mM EDTA, 2 mM dithiothreitol and 50 mM KC1. The suspension was strained through four layers of cheese cloth and the extract retained. The pulp was homogenized again for 1 min with another 100 ml portion of extraction buffer. The extract obtained after straining the second homogenate, was combined with the first filtrate and clarified by 15-min centrifugation at 1 2 0 0 0 X g at 4°C. The solution obtained was fractionated by addition of solid ammonium sulfate, or by addition of solid poly(ethylene glycol) M r 6000. Precipitates obtained by these steps were collected by centrifugation at 12000 x g for 20 min. These were dissolved in 20 ml of the extraction buffer, and the solutions were dialyzed overnight against 100 volumes of extraction buffer at 4°C. The dialysis was required to remove ketosyl compounds (mostly sucrose) that could significantly interfere in the resorcinol reaction. This exhaustive dialysis of enzyme samples was not obligatory when the assays of enzymatic activities were based on the spectrophotometric coupled enzyme reactions as described above. Results

Activities of both 6-phosphofructo-2-kinase as well as of fructose-2,6-bisphosphatase were present in the mung bean seedlings extracts (Table I). The total activity of the 2-kinase is of the same order of magnitude as that of the classical 6-phosphofructose-l-kinase and that of the PPi:fructose-6-phosphate-l-phosphotransferase, all three enzymes which compete for the same substrate, fructose-6-P. Similarly, the level of activity of the fructose-2,6bisphosphatase is in the same range found for fructose-l,6-bisphosphatase. Nearly all of these enzymic activities could be recovered in the

320 TABLE I E N Z Y M I C ACTIVITIES IN SEEDLINGS E X T R A C T S Activities expressed in n m o l / m g protein per rain using the assay conditions described in Methods and Materials. Protein values represent the yield from a 100 g batch of sprouts. Enzyme

Crude extract

(N H 2 ) z So4 fraction (20-50%)

Poly(ethylene glycol) fraction (0-6%)

6-Phosphofructo-l-kinase 6-Phosphofructo-2-kinase PPi:6-phosphofructo-l-phosphotransferase Fructose-l,6-bisphosphatase Fructose-2,6-bisphosphatase Total protein (yield) (mg)

64.8 a; 40.2 19.5; 60.0 ~ 35.8 3.5 9.9 278

82.2 4; 60.4 23.9; 89.0 ~ 37.1 17.5 16.3 97

63.1 a; 67.9 47.3; 68.0 b 56.7 12.2 10.4 102

In the presence of added 0.5 m M AMP. b In the presence of added 5 m M Pi.

20--50% ammonium sulfate fraction, as well as in the fraction precipitated by addition of poly(ethylene glycol) to a final concentration of 6%. Further experiments to purify both the 6-phosphofructo2-kinase and the fructose-2,6-bisphosphatase free from nonspecific contaminating enzymic activities and to precisely characterize their substrate specificity are being pursued in our laboratory. In absence of added orthophosphate the 6phosphofructo-2-kinase shows a typical sigmoidal curve when dependence of activity is measured against increasing concentrations of either ATP or fructose-6-P as the substrates (Fig. 1). Addition of inorganic phosphate strongly increased rates of kinase activity resulting in a typical hyperbolic

curve towards both of these substrates. Conversely, addition of 3-phosphoglycerate had a small but significant inhibitory effect on the reaction. Whereas g m for fructose-6-P and ATP were 0,4 and 0,2 mM respectively in presence of P~, the apparent So.5 values were elevated to 1.6 and 1.2 mM in absence of added Pi, and to 1.8 and 1.5 mM in presence of 3-phosphoglycerate. It is suspected that the true So.5 values in absence of Pi are higher than the values recorded above because the reagents used for the assays contaminated the test system with about 10-40/~M P~. It was also possible that some phosphatase activities in the impure enzyme preparations could release Pi (particularly from the ATP) during the incubation period. Vmax

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of the reaction increased only modestly in the presence of P~, and maximal activation occurred at about 4 mM Pi (Fig. 1C). Activation declines slightly at Pi concentrations above 6 mM. Addition of 3-phosphoglycerate could not completely abolish the activation effect of Pi (Fig. 1C). 1-2 m M 3-phosphoglycerate inhibited only 20-50% of the maximal activity of the enzyme which was activated by 5 mM Pi. Fructose-2,6-bisphosphatase activity was sig-

nificantly inhibited by addition of orthophosphate (10.5 at about 0.8 mM), but only moderately activated by 3-phosphoglycerate (Ao. 5 at about 0.3 mm; Fig. 2). K m for fructose-2,6-P2 which was 0.38 mM in absence of Pi changed to 1.4 mM when Pi was present. Apparently the same K m value (0.36 mM) as in the absence of Pi was found when 3-phosphoglycerate was added. The double reciprocal plot (Fig. 2) indicated that there is a non-competitive type of relationship between fructose-2,6-P2 and the two effectors studied. The results suggest that a competition between P~ and 3-phosphoglycerate occurs, but unfortunately a detailed kinetic analysis of this relationship did not yield data which was sufficiently accurate to define it more precisely. Elimination of Mg 2+ from the reaction mixture has resulted in a sharp decline (70-80%) in fructose-2,6-bisphosphatase activity indicating a strong dependence on this divalent cation for catalytic activity. Substitution of 3-phosphoglycerate with 2 mM phosphoenolpyruvate has shown a much weaker but similar effect as 3-phosphoglycerate on the activities of both the fructose-6-P-2-kinase and the fructose2,6-bisphosphatase activity examined (results not shown). It is also hoped that further studies with a preparation of fructose 1,2-bisphosphatase clear of any contaminating unrelated phosphatase activities will provide us with further detailed kinetic data on the regulatory properties of this enzyme. Discussion The presence of 6-phosphofructo-2-kinase and the fructose-2,6-bisphosphatase activities in the mung bean seedlings extracts is at the same high level found for some other well known related enzymes present in the same extracts (Table I). This is in contrast with mammalian tissues where total activity of these two enzymes is comparatively low [1-3,14-18]. This finding means that the three phosphorylating enzymes (6-phosphofructo1-kinase, 6-phosphofructo-2-kinase and the PPi:6phosphofructo-l:phosphotransferase) are very effective competitors for the pool of fructose-6-P present in the cytoplasm. Selective modulation of the activities of these three enzymes as well as the availability of the co-substrates ATP and PPi, will therefore determine the relative dominance of a

322

particular fructose-6-P utilizing reaction at specific physiological and metabolic states. For example, increased levels of Pi could accelerate production of fructose-2,6-P2, which in turn will activate the PPi:6-phosphofructo-l-phosphotransferase reaction. But at the same time, if PP~ levels are very low, this elevation of Pi may favor formation of fructose-6-P from fructose-l,6-P2. Increase in 3phosphoglycerate will counteract some of the modulating effects of Pi and probably will result in diminished intercellular fructose-2,6-P2 levels. While it is difficult to evaluate how the balance of these opposing or parallel effects on individual reactions will change the flow of metabolic carbons in the non-photosynthetic tissue, it is interesting that results obtained with photosynthetic tissue indicate similar activation/inhibition patterns [19]. In our laboratory we could confirm the presence of 6-phosphofructo-2-kinase (23.1 n m o l / m i n per mg protein) and fructose-2,6-bisphosphatase (13.7 n m o l / m i n per mg protein) in spinach-leaf extracts (Avigad, G., Bohrer, P.J. and Nadler, S., unpublished data). Only further studies could establish whether the plant enzymes are similar in molecular character to the bifunctional-6-phosphofructo-2kinase/fructose-2,6-bisphosphatase protein isolated from mammalian liver [14-18]. The central role of Pi and 3-phosphoglycerate levels in controlling fluxes of metabolic carbons in such key processes as starch biosynthesis, export of photosynthate from the plastids, glycolysis and supply of carbons for the biosynthesis of sucrose is well established [25-31]. The observation that the same pair of effectors can also exert a strong control on a pair of enzymes associated with fructose-2,6-P2 biosynthesis and degradation is of prime interest. The suggested central position of fructose-2,6-P2 in the flow of carbohydrates through the major metabolic pathways in the growing etiolated seedling is illustrated in Fig. 3. The overwhelming metabolic processes which operate in the germinating legume seed involve the mobilization of storage materials (e.g., galactomannan, starch, sucrose oligosaccharides, and triglycerides) to provide building blocks for the biosynthesis of major products such as sucrose for transport and various structural elements, such as cell wall glycosides, in the growing tissues. A certain portion of the supply

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of monosaccharides provided by the hydrolysis of storage glycosides, as well as some of the products obtained from triglyceride catabolism, have to be utilized through glycolysis and the oxidative pathways to secure the generation of the large amount of ATP required for the biosynthetic processes. The dynamics of the cycle which exists between fructose-6-P and fructose-l,6-P2 reflects the balance between the fluxes of carbohydrates channelled towards the biosynthetic sinks, and that diverted for energy production. Inorganic phosphate and 3-phosphoglycerate directly or by controlling the level of fructose-2,6-P2, will modulate the flow of carbohydrates through this metabolic juncture. A significant amount of PP~ produced during the formation of the pool of nucleotide sugars [32] is available for the synthesis of fructose-l,6-P2 by the fructose-2,6-P2 controlled PPi: fructose-6-P-l-phosphotransferase reaction. However, since pyrophosphatases abound in the plant tissue, activation of this reversible phosphotransferase reaction can rather lead to the conversion of fructose-l,6-P2 back to fructose-6-P. It is however difficult without further studies to evaluate the quantitative contribution of this PPi:phosphotransferase reation to the overall fructose-l,6-P2/fructose-6-P cycling patterns in

323

this tissue. It is interesting to note that the one indirect channel available for conversion of fructose-6-P to fructose-l,6-P 2 and which is not subjected to control by fructose-2,6-P 2 or by Pi is via the hexose monophosphate pathway. In this bypass fructose-l,6-P 2 could be obtained from glyceraldehyde 3-phosphate molecules formed in the transaldolase and transketolase reactions. This channel can also contribute to the synthesis of some fructose-6-P from the tricarbon intermediates generated from the catabolism of triglycerides. The information available at present about the fluctuations in intracellular fructose-2,6-P 2 concentrations during different physiological states, and their relationship to the concentrations of Pi, 3-phosphoglycerate and other metabolites in the same subcellular metabolic compartment, is as yet very limited. It is however clear that the concentration of this bisphosphate in the developing seedling tissue studied here is at high levels which can maximally enhance PP:fructose-6-P-l-phosphotranferase or reduce fructose-l,6-bisphosphatase activities in vitro [6,10]. It is conceivable that in vivo these effects do not reach the extreme magnitudes which can be simulated in the test-tube because of the contributing effects of many other modulators. As a speculative suggestion, it should be considered that fructose-2,6-P 2 is not only a compound with an exclusive metabolic role as a regulator of key enzymic reactions, as it has been suggested for mammalian tissues. It may also be an intermediate in as yet unrecognized biochemical reactions. For example, being a glycoside, fructose-2,6-P 2 could be involved in some glycosylation reactions. It should be recalled that nucleoside diphosphate fructose derivatives were in the past isolated from plant tissues but their metabolic role remains a mystery [32]. A possible link between these compounds and fructose-2,6-P 2 should be explored. Two recent reports [33,34] add further support to the suggestion that levels of fructose-2,6-P 2 contribute to the regulation of carbon flow in plant tissues. These studies [34] also confirm the presence in spinach leaves of a fructose-2,6-bisphosphatase with some regulatory properties similar to those noticed for the mung bean enzyme described in our own experiments.

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32 Feingold, D.S. and Avigad, G. (1980) m l h e Biochemistry of Plants, Vol. 3 (Preiss, J., ed.), pp. 101-170, Academic Press, New York 33 Stitt, M., Gerhardt, R., Kiarzel, B., and Heldt, H.W. (1983) Plant Physiol. 72, 1139-1141 34 Csrke, C., Stitt, M., Balogh, A. and Buchanan, B.B. (1983) FEBS Lett. 162, 103-106