Mar 5, 1984 - ... Sucrose Phosphate. Synthase as Affected byGlucose-6-Phosphate and .... contained 50 mm Hepes-NaOH (pH 7.0), 5 mM MgCl2, plus substrates (F6P and ... A, F6P concentration versus SPS activity; B, Woolf plot of data.
Plant Physiol. (1984) 76, 250-253 0032-0889/84/76/0250/04/$0 1.00/0
Phosphate Inhibition of Spinach Leaf Sucrose Phosphate Synthase as Affected by Glucose-6-Phosphate and Phosphoglucoisomerasel Received for publication March 5, 1984 and in revised form May 24, 1984
DOUGLAS C. DOEHLERT* AND STEVEN C. HUBER United States Department ofAgriculture, Agricultural Research Service, and Departments of Botany and Crop Science, North Carolina State University, Raleigh, North Carolina 27695-7631 ABSTRACI The inhibition patterns of inorganic phosphate (Pi) on sucrose phosphate synthase activity in the presence and absence of the allosteric activator glucose-6-P was studied, as well as the effects of phosphoglucoisomerase on fructose-6-P saturation kinetics with and without Pi. In the presence of S millimolar glucose-6-P, Pi was a partial competitive inhibitor with respect to both substrates, fructose-6-P and uridine diphosphate glucose. In the absence of glucose-6-P, the inhibition patterns were more complex, apparently because of the interaction of Pi at the activation site as well as the catalytic site. In addition, substrate activation by uridine diphosphate glucose was observed in the absence of effectors. The results suggested that Pi antagonizes glucose-6-P activation of sucrose phosphate synthase by competing with the activator for binding to the modifier site. The fructose-6-P saturation kinetics were hyperbolic in the absence of phosphoglucoisomerase activity, but became sigmoidal by the addition of excess phosphoglucoisomerase. The sigmoidicity persisted in the presence of Pi, but sucrose phosphate synthase activity was decreased. The apparent sigmoidal response may represent the physiological response of sucrose phosphate synthase to a change in hexose-P concentration because sucrose phosphate synthase operates in the cytosol in the presence of high activities of phosphoglucoisomerase. Thus, the enzymic production of an activator from a substrate represents a unique mechanism for generating sigmoidal enzyme kinetics.
SpS2 (EC 2.4.1.14) is an enzyme of central importance to the process of sucrose biosynthesis. It catalyzes the synthesis of sucrose-P from F6P and UDPG. Results from this laboratory have indicated that spinach leaf SPS is activated by G6P and that this activation is antagonized by Pi (6, 7). We hypothesized a regulatory mechanism whereby an abundance of G6P would indicate to the cell that sufficient fixed carbon was available for export, thus activating sucrose synthesis. Similarly, an increase in Pi might correspond to a decreased rate of Pi/triose-P exchange from the chloroplast and a decrease in cystolic sugar phosphates.
Thus, the G6P/Pi ratio could provide a mechanism for the metabolic fine control of SPS activity based on the supply of reduced carbon from the chloroplast. One aspect of SPS regulation that we did not consider earlier is the role of PGI, which would maintain an equilibrium mixture of F6P and G6P in the cytosol with a mass-action ratio of 0.22 to 1 (3). Because spinach leaf cytosol contains high PGI activity (1 1), the response of SPS activity to an equilibrium mixture of F6P and G6P, catalyzed by PGI, might better reflect how changes in substrate availability in situ might affect SPS activity. An additional aspect of SPS regulation that required further clarification was the pattern of Pi inhibition of SPS in the presence and absence of G6P. Earlier studies of Pi inhibition of SPS were either performed on SPS preparations contaminated with PGI activity, or did not consider the effect of G6P on the pattern ofinhibition. Consequently, the specific objectives of this study were to determine (a) the inhibition kinetics of Pi on SPS in the presence and absence of the activator G6P and (b) the effects of PGI on F6P saturation kinetics in the presence and absence of Pi.
MATERIALS AND METHODS Spinach (Spinacia oleracea L.) was obtained from a local market. SPS was extracted and partially purified as described in detail elsewhere (6, 7) involving chromatography on the hydrophobic resin, w-aminohexyl-agarose (8), and gel filtration on Ultrogel AcA 34.3 The final preparation was free of contamination activities of PGI, phosphoglucomutase, UDPG epimerase, and nonspecific phosphatases. SPS activity was assayed by fixed point (10 min) determination of sucrose phosphate production by the method of Cardini et al. (4, 9). The assay mixture (70 11) contained 50 mm Hepes-NaOH (pH 7.0), 5 mM MgCl2, plus substrates (F6P and UDPG) and effectors (G6P and Pi) as indicated. Blank reactions contained the complete assay plus denatured enzyme. One unit of SPS activity is defined as the amount of enzyme necessary to produce one nmol of sucrose-P in 1 min at 30°C, pH 7.0. The straight lines shown in doublereciprocal and Woolf plots were obtained by linear regression of the data. For assays containing PGI, approximately 0.25 IU of commercially obtained (Sigma) PGI was added to each reaction mixture approximately 5 min before initiating reactions by addition of SPS.
' Cooperative investigations of the Agricultural Research Service, United States Department of Agriculture and the North Carolina Agricultural Research Service, Raleigh, NC. Paper No. 8972 of the Journal series of the North Carolina Agricultural Research Service, Raleigh, NC 3Mention of a trademark or proprietary product does not constitute 27695-7631. 2 Abbreviations: SPS, sucrose phosphate synthase; F6P, fructose-6-P; a guarantee or warranty of the product of the United States Department UDPG, uridine diphosphate glucose; G6P, glucose-6-P; PGI, phospho- of Agriculture or the North Carolina Agricultural Research Service and glucoisomerase; A3o (G6P), concentration of G6P necessary for half- does not imply its approval to the exclusion of other products that may also be suitable. maximal activation. 250
PHOSPHATE INHIBITION OF SUCROSE PHOSPHATE SYNTHASE
251
RESULTS Effects of Anions. Phosphate inhibition of SPS activity has been well documented (2, 6-8). Table I shows that sulfate, like Table I. Effects of Various Anions on SPS Activity Assays were run with 8 mM F6P and 8 mM UDPG at pH 7.0. Each value is the mean of two determinations. SPS Activity in Presence of Anion Concn Phosphate Sulfate Arsenate % control rate mM 87 100 5 58 100 10 40 59 20 43 56 101
[F6P],mM
[F6P], mM
4/[F6P], mM
'
FIG. 1. Double reciprocal plots of the effect of Pi on F6P saturation kinetics in the presence of 5 mm G6P. All assays contained 8 mM UDPG at pH 7.0.
I /[UDPG], mM '
FIG. 2. Double reciprocal plots of the effect of Pi on UDPG saturation kinetics in the presence of 5 mM G6P. All assays contained 8 mM F6P at pH 7.0.
FIG. 3. Effect of Pi on F6P saturation kinetics in the absence of G6P. All assays contained 8 mM UDPG at pH 7.0. A, F6P concentration versus SPS activity; B, Woolf plot of data.
Pi, was also an effective inhibitor whereas arsenate, another phosphate analog, was not inhibitory. It is surprising that arsenate does not inhibit spinach leaf SPS, since the ability to differentiate between phosphate and arsenate is rare in enzyme systems. Table I also documents that Pi (and sulfate) inhibition was partial. Little or no further inhibition of enzyme activity was observed when the concentration of Pi was increased above 20 mm (data not shown). Pi Inhibition with G6P. In the presence of 5 mM G6P, Pi was a competitive inhibitor with respect to both F6P (Fig. 1) and UDPG (Fig. 2). Because Pi inhibition was not complete, Pi must be classified as a partial competitive inhibitor with respect to both substrates in the presence of G6P. Pi Inhibition without G6P. In the absence of G6P, the inhibition patterns of Pi on substrate saturation were more complex. The effect of increasing Pi concentration on F6P saturation kinetics in the absence of G6P is shown in Figure 3A and Woolf plots of the data are shown in Figure 3B. The response of SPS to increasing F6P was hyperbolic and the Woolf plots were linear, but the lines intersected to the right of the abscissa. The apparent Km (F6P) in the absence of G6P was 3.2 mm. As the concentration of Pi was increased, the Km (F6P) decreased as did the apparent Vm,,. Double reciprocal plots on the data were curved and tended to flatten out at low substrate concentration (not shown). This effect seemed amplified by the bias of double reciprocal plots for low substrate concentration points. It appears that in the absence of G6P, Pi can interact as a weak activator resulting in a lowering of the apparent Km (F6P), but also inhibits and lowers the V,,,. According to Segal (12), this pattern is produced by a 'mixed-
252
DOEHLERT AND HUBER
type inhibitor.' In addition, competitive effects of Pi at the catalytic site may contribute to this overall effect. The effects of Pi on UDPG saturation kinetics were also complex in the absence of G6P (Fig. 4A). In the absence of Pi, double reciprocal plots of UDPG saturation kinetics were biphasic (Fig. 4B). At low UDPG concentrations (0 to 8 mM), SPS had an apparent Km (UDPG) of 1.8 mM and a V. of 107 units/ ml. At higher UDPG concentrations (8 to 32 mM), the apparent Km (UDPG) was 9.5 mM and the calculated V. was 301 units/ ml. The biphasic response is suggestive of substrate activation (5) and could indicate that UDPG, at high concentrations, can interact at the activation site. Substrate activation was not apparent with F6P (Fig. 1) or with UDPG in the presence of 5 mM G6P (Fig. 2). Increasing the Pi concentration increased the slope of the double reciprocal plot especially at low substrate concentrations so that all the lines converged at about the same point on the vertical axis (Fig. 4B). The presence of Pi also reduced
substrate activation and the double reciprocal plots appeared linear. Greater inhibition of SPS by Pi at low UDPG concentrations suggested a strong competitive component, like that seen in the presence of G6P. Effects of PGI on F6P Saturation Kinetics. In the absence of PGI, the response of SPS to increasing F6P concentration was
|2 9 mM 5.5mM
240 220-
+PGI
70
_ 60 640 -
/
, |
° D 40
O
-PGi
/ 0TUALCEP].. / 2
/
30
-
/ >
'
-
/
"co
_
0
,
2
3
INITIAL
4
5
[6P], mM
6
7
8
FIG. 5. Effect of PGI (3.5 IU/ml) on F6P saturation kinetics of SPS. Inset, plot of +PGI curve according to the calculated F6P concentration in the reaction mixture, assuming an equilibrium mixture of 0.22: 1, F6P to G6P. The response of SPS without PGI is plotted to the same range of F6P concentrations.
280
260260 0
Plant Physiol. Vol. 76, 1984
0//V lOmM
hyperbolic (Figs. 1, 5). When a high activity of PGI (3.5 IU/ml)
200
was added to the reaction
mixtures before assay, the response became sigmoidal (Fig. 5). The excess PGI added would maintain 160 / /%V/ _ an equilibrium mixture of F6P (substrate) and G6P (activator) 140 / 2,!// _ in a ratio of 0.22 to 1 (3), thus substantially decreasing the actual 120 f /// _ F6P concentration. At low initial F6P, the presence of G6P does / /// not sufficiently activate the enzyme to make up for the decrease in activity due to reduction in F6P concentration; thus, the SPS 80 _ /// _ activity is lower with PGI than in the control. With increased initial F6P the combination of increased substrate and the in60 - / D/ concentration ofactivator results in SPS activities higher creased 40 _cZ / _ than those without PGI. This phenomenon of proportionally increasing activator with increasing substrate is apparently re4 24 28 32 8 16 20 12 sponsible for the generation of sigmoidal kinetics. As shown in Figure 5, when the initial concentration of F6P [UDPG] mM was low (up to 2.5 mM), addition of PGI resulted in lower SPS activities relative to the control (minus PGI). However, when 016 015 SPS activity was plotted as a function of actual F6P concentrait isinclear that the G6P / X tion, generated by the added PGI5, always 0.14 /5 resulted a substantial activation of SPS activity (Fig. inset). 013 _ In the presence of PGI, SPS activity increased sigmoidally in 0.12 _ mresponse to actual F6P concentration because the concentration 010, of the activator, G6P, was also increasing. Pi Inhibition in the Presence of PGI. The effect of Pi on the 0.09 apparent F6P saturation kinetics of SPS in the presence of PGI / /are presented in Figure 6. In the absence of Pi, SPS activity 00700-//.mm _ incrsed sigmoidally as the initial F6P concentration was in0.06 creased. In the presence of Pi, the apparent S5o was increased 0.05 and the maximal velocity was decreased. Apparent sigmoidal 004 kinetics were observed in the presence of 2.5 and 5 mM Pi, but No at 10 mm Pi the activity curve began to appear hyperbolic. 003 >
20
-
002
00'
DISCUSSION It appears from these results that Pi can interact with SPS at two distinct sites, the catalytic site and an activation site. In the I/[UOP(limMFIG. 4. Effect of Pi on UDPG saturation kinetics in the absence of presence of the allosteric activator, G6P, Pi appeared to act G6P. All assays contained 8 mm F6P at pH 7.0. A, UDPG concentration strictly at the catalytic site and was a competitive inhibitor for versus SPS activity plot; B, double reciprocal plot of data. both UDPG and F6P (Figs. 1, 2). In the absence of G6P, Pi 05
1.0
i5
20
PHOSPHATE INHIBITION OF SUICROSE PHOSPHATE SYNTHASE
253
increases the Km (UDPG). Our observations are also consistent with those of Amir and Preiss (2) who found that Pi increased the S5o (F6P) in the presence of PGI. However, our observation that Pi was competitive with F6P (in the presence of G6P) is contradictory to several reports (8, 10), and the differing results ltoo cannot be easily reconciled. It is not surprising that Pi would be a competitive inhibitor for both F6P and UDPG since they are ~80 both phosphate esters. The interaction of Pi with G6P activation has been characterized previously (6, 7). The absence of G6P results in complex patterns of Pi inhibition. The presence of G6P simplifies the response by apparently eliminating the interaction of Pi with the activation site. The failure of earlier workers to report these patterns and G6P activation may be attributed to contamination of their preparations with trace activities of PGI. Our results suggest that Pi can interact at three separate sites200 at the UDPG binding site, the F6P binding site, and at the G6P activation site. These interactions along with UDPG binding at the activation site (i.e. substrate activation), apparently produce the complex Pi inhibition patterns observed. 8 10 12 4 6 2 The activity of SPS in situ will depend on the concentrations of substrates, F6P and UDPG, and metabolic effectors, G6P and INITIAL [F6P], mM Pi. The concentrations of these metabolites in the cytosol, as well FIG. 6. Effect of Pi on F6P saturation kinetics in the presence of PGI. as the rate of sucrose formation, has been shown to vary with photosynthetic rate. In studies with spinach photoplasts, Stitt et inhibition patterns became more complex, which we attribute to al. ( 13) manipulated photosynthetic rates by changing either light the interaction of Pi at the activation site. Interaction of Pi at intensity or CO2 concentration. As the rate of carbon fixation the activation site (in the absence of G6P) apparently caused increased, the concentration of P-esters in the cytosol increased enough of a conformational change to lower the Km (F6P). G6P as did the rate of carbon flux into sucrose. Stitt et al. (13) noted activation lowers the Km (F6P) from about 3.0 to 0.6 mm, but Pi that hexose-P (F6P + G6P) was the principal component of antagonizes this activation by increasing the Aso (G6P) from 0.85 cytosolic P-esters to change in concentration. How SPS activity mM with no Pi to 9.8 mm with 20 mM Pi (7). The mechanism of in situ might respond to these changes in metabolite concentrathis antagonism may be the displacement of G6P from the tions may be approximated by the data presented in Figure 6, activation site by Pi. Additional evidence for this hypothesis is where F6P saturation kinetics were performed in the presence of that Pi eliminated substrate activation by UDPG (Fig. 4). In this excess PGI activity. As shown, SPS activity increased sigmoidally case, Pi would be displacing UDPG from the activator site. rather than hyperbolically. Thus, changes in metabolite concenApparently, at low F6P, the activation effect of Pi masks the trations in the cytosol that occur with changes in photosynthetic competitive inhibition component of Pi with F6P, because little rate could have dramatic effects on the rate of sucrose biosyninhibition was observed under these conditions. thesis. Substrate activation by UDPG, which has not been reported LITERATURE CITED previously, was apparent from the biphasic nature of the double reciprocal plots (Fig. 4B). It is of interest to note that Harbron et 1. AKAZAWA T, K OKAMOTO 1980 Biosynthesis and metabolism of sucrose. In J Preiss, ed, Biochemistry of Plants, Vol 3. Academic Press, New York, pp al. (8) reported a Km (UDPG) of 9.5 mm, which is consistent 199-220 with the higher concentration range Km (UDPG) that we have 2. AMIR J, J PREISS 1982 Kinetic characterization of spinach leaf sucrose-phosattributed to substrate activation. However, this phenomenon phate synthase. Plant Physiol 69: 1027-1030 because it may be of limited physiological significance appears 3. AP REEs T 1980 Integration of pathways of synthesis and degradation of hexose phosphates. In J Preiss, ed, Biochemistry of Plants, Vol 3. Academic Press, that the concentration of UDPG in the leaf cell rarely exceeds 2 New York, pp 1-42 mM (13). CE, LF LELOIR, J CHIRIBOGA 1955 The biosynthesis of sucrose. J Biol The dramatic effects of G6P on Pi inhibition emphasize the 4. CARDINI Chem 214: 149-155 all importance of eliminating phosphoglucoisomerase activity 5. DiXON M, EC WEBB 1979 Enzymes. Academic Press, New York, p 138 from SPS preparations intended for kinetic analysis because PGI 6. DOEHLERT DC, SC HUBER 1983 Spinach leaf sucrose phosphate synthase: Activation by glucose-6-phosphate and interaction with inorganic phosphate. will convert a substrate, F6P, into an activator, G6P. The data FEBS Lett 153: 293-296 presented in Figure 5 verify that addition of excess PGI to 7. DOEHLERT DC, SC HUBER 1983 Regulation of spinach leaf sucrose phosphate F6P saturation kinetics from SPS can convert purified (PGI-free) synthase by glucose-6-phosphate, inorganic phosphate and pH. Plant Physiol 73: 989-994 hyperbolic to sigmoidal. A similar sigmoidal response to F6P 8. HARBRON SC, C FOYER, D WALKER 1981 The purification of sucrose phosphate was observed by Amir and Preiss (2) with spinach leaf SPS. synthase from spinach leaves: The involvement of this enzyme and fructose Although their enzyme preparation contained PGI, they attribbisphosphatase in the regulation of sucrose biosynthesis. Arch Biochem SPS for kinetics to of uted the sigmoidal positive cooperativity Biophys 212: 237-246 F6P. The persistence of hyperbolic kinetics in the absence of 9. HUBER SC 1981 Interspecific variation and regulation ofleaf sucrose phosphate synthase. Z Pflanzenphysiol 102: 443-450 PGI excludes cooperativity as a mechanism for generating the GL, HG PONTIS 1977 Studies on the sucrose phosphate synthetase sigmoidal response. The presence or absence of PGI in SPS 10. SALERNO kinetic mechanism. Arch Biochem Biophys 180: 298-302 preparations may also be the reason for the wide range of 11. SCHNARRENBERGER K, A OESER 1979 Two isozymes of glucosephosphate isomerase from spinach leaves and their intracellular compartmentation. apparently conflicting reports of sigmoidal versus hyperbolic Eur J Biochem 45: 77-82 saturation kinetics for F6P (see Ref. 1). SEGAL IH 1975 Enzyme Kinetics. John Wiley & Sons, New York, pp 189-192 All earlier studies have reported that Pi inhibition was com- 12. 13. STITT M. W WIRTZ, HW HELDT 1983 Regulation of sucrose synthesis by petitive with UDPG and noncompetitive with F6P (2, 8, 10). cytoplasmic fructose bisphosphatase and sucrose phosphate synthase in Pi in varying light and carbon dioxide. Plant Physiol 72: 767-774 Our results are consistent with the previous reports that w -J
