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Keywords:

  • metabolic control analysis;
  • sugarcane;
  • sucrose synthase;
  • kinetic modelling

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Enzyme purification and chromatography
  6. SuSy assays
  7. Determination of kinetic parameters and modelling
  8. Results
  9. Primary (Hanes–Woolf) plot analysis
  10. Product inhibition studies
  11. Modelling
  12. Discussion
  13. Acknowledgements
  14. References

The kinetic data on sugarcane (Saccharum spp. hybrids) sucrose synthase (SuSy, UDP-glucose: d-fructose 2-α-d-glucosyltransferase, EC 2.4.1.13) are limited. We characterized kinetically a SuSy activity partially purified from sugarcane variety N19 leaf roll tissue. Primary plot analysis and product inhibition studies showed that a compulsory order ternary complex mechanism is followed, with UDP binding first and UDP-glucose dissociating last from the enzyme. Product inhibition studies showed that UDP-glucose is a competitive inhibitor with respect to UDP and a mixed inhibitor with respect to sucrose. Fructose is a mixed inhibitor with regard to both sucrose and UDP. Kinetic constants are as follows: Km values (mm, ± SE) were, for sucrose, 35.9 ± 2.3; for UDP, 0.00191 ± 0.00019; for UDP-glucose, 0.234 ± 0.025 and for fructose, 6.49 ± 0.61. inline image values were, for sucrose, 227 mm; for UDP, 0.086 mm; for UDP-glucose, 0.104; and for fructose, 2.23 mm. Replacing estimated kinetic parameters of SuSy in a kinetic model of sucrose accumulation with experimentally determined parameters of the partially purified isoform had significant effects on model outputs, with a 41% increase in sucrose concentration and 7.5-fold reduction in fructose the most notable. Of the metabolites included in the model, fructose concentration was most affected by changes in SuSy activity: doubling and halving of SuSy activity reduced and increased the steady-state fructose concentration by about 42 and 140%, respectively. It is concluded that different isoforms of SuSy could have significant differential effects on metabolite concentrations in vivo, therefore impacting on metabolic regulation.

Abbreviations
MCA

metabolic control analysis

SuSy

sucrose synthase

The kinetic parameters of enzymes provide important information about their interactions with substrates, products and effectors. Typically, substrate Km values are interpreted to give an indication of the affinity of enzymes for their substrates, and conclusions about enzymes' physiological roles are often based on these values. However, the kinetic parameters of individual enzymes do not by themselves provide much insight into the behaviour of an intact, functioning metabolic pathway. Cellular network models, such as those applied in the approach of computational systems biology, extend the usefulness of kinetic data on individual enzymes immensely and can have both explanatory and predictive value.

Several papers that give an overview of different approaches for studying and modelling metabolism, such as metabolic flux analysis, metabolic control analysis (MCA) and positional isotopic labelling combined with NMR or MS, have been published recently [1–3]. Of these approaches, MCA [4,5] is particularly useful in studies of metabolic pathways, as it quantifies the degree of control of individual reaction steps on the steady-state pathway flux or metabolite concentrations. Hence, MCA can be a great help in determining potential target steps for metabolic engineering, because the reactions in the pathway that have the most potential of modifying a target flux or metabolite concentration can be identified. For example, MCA has been used to study the control of different steps on mitochondrial respiration [6], and successfully predicted that overexpression of NADH oxidase is more successful than acetolactate synthase overexpression for increasing production of diacetyl by Lactococcus lactis[7]. In plants, MCA was used to estimate the flux control coefficient of phosphoglucoisomerase on sucrose and starch production using Clarkia xantiana mutants with decreased levels of this enzyme [8]. MCA has been discussed in the context of plant metabolism [9] and further examples of its application are given therein, as well as practical advice on isolation and assay of plant enzymes and extraction of metabolites. It should be mentioned that plants pose particular challenges as far as analysis of their metabolism by MCA (or other methods for that matter) is concerned: the degree of compartmentalization of metabolism is extremely high, and isolation of active enzymes can be a challenge, owing to various factors such as proteases, interfering compounds, high acidity and so forth. Apart from these considerations, the lack of uniform data sets for use in the construction of kinetic models can be a hindrance. Addressing this point, techniques to measure considerable numbers of metabolites simultaneously are now available and will contribute greatly to analyses of metabolism and our understanding thereof [10].

A kinetic model describing sucrose accumulation in sugarcane was published recently [11]. This model was used to calculate the control coefficients of enzymes in the sucrose synthesis pathway for sucrose futile cycling (cleavage and resynthesis of sucrose), with a view to determining which reactions control this energetically wasteful process. Like any kinetic model, it requires the rate equations of all reactions in the pathway and therefore the kinetic parameters of every enzyme. Typically the rate equations require more information than simply Km values for the substrates, which are the only kinetic parameters reported in most studies not focusing exclusively on kinetics. For sugarcane SuSy (SuSy, UDP-glucose: d-fructose 2-α-d-glucosyltransferase, EC 2.4.1.13), substrate Km values have been reported [12], but not other important parameters, such as substrate Ki values, or confirmation of the reaction mechanism, which are also needed for kinetic modelling.

The objective of this study was to obtain more extensive data on the kinetic parameters of sugarcane SuSy, which can be used to enhance modelling of sucrose accumulation and also improve our understanding of sugarcane SuSy and its influence on sucrose accumulation.

Materials

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Enzyme purification and chromatography
  6. SuSy assays
  7. Determination of kinetic parameters and modelling
  8. Results
  9. Primary (Hanes–Woolf) plot analysis
  10. Product inhibition studies
  11. Modelling
  12. Discussion
  13. Acknowledgements
  14. References

Sugarcane (Saccharum spp. hybrids), variety N19, field grown at the University of Stellenbosch experimental farm was used. Internode one was taken as the internode attached to the leaf with the first exposed dewlap [13].

Tris buffer, dithiothreitol and all coupling enzymes were obtained from Roche (Basel, Switzerland), except UDP-glucose pyrophosphorylase, which was from Sigma (3050 Spruce St., St. Louis, MO, USA). Merck (Darmstadt, Germany) provided the other chemicals.

Enzyme purification and chromatography

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Enzyme purification and chromatography
  6. SuSy assays
  7. Determination of kinetic parameters and modelling
  8. Results
  9. Primary (Hanes–Woolf) plot analysis
  10. Product inhibition studies
  11. Modelling
  12. Discussion
  13. Acknowledgements
  14. References

Leaf roll tissue was ground to powder in liquid nitrogen and extracted in a 1 : 2 (m/v) ratio of 300 mm Tris/HCl (pH 7.5) buffer containing 10% (v/v) glycerol, 2 mm MgCl2, 5 mm dithiothreitol, 2 mm EDTA and Roche Complete™ protease inhibitor. The homogenate was filtered through a double-layered nylon cloth, centrifuged at 10 000 g for 10 min, and the pellets discarded. The proteins in the supernatant were precipitated by 80% saturation with ammonium sulfate and recovered by centrifugation at 10 000 g for 10 min. The pellets were resuspended in 100 mm Tris/HCl (pH 7.5) buffer containing 2 mm MgCl2, 2 mm dithiothreitol and 2 mm EDTA (buffer A). The protein extract was then desalted by passage through a Pharmacia PD-10 (Sephadex G25) column and the eluant was diluted two times with buffer A. The desalted extract was applied to a 5 mL Amersham/Pharmacia Hi-trap Q anion exchange column that had previously been equilibrated with buffer A. The protein was eluted with a linear KCl gradient at a flow speed of 1 mL·min−1 and fractions containing 20% or more of maximum activity were pooled. Active fractions from the column were dialysed against buffer A.

The partially purified extract was tested for the potential presence of the interfering activities invertase, UDPGlc dehydrogenase, fructokinase and sucrose phosphate synthase. Results showed that under the conditions used for the SuSy assays (pH 7 for the sucrose breakdown assay or pH 7.3 for the synthesis reaction, 100 mm Tris buffer) there were no significant levels of these interfering activities present, with only invertase barely detectable at less than 0.5% of SuSy activity. This partially purified SuSy activity (named SuSyC) was one of three SuSy activities in leaf roll which differed in their chromatographic, kinetic and immunological properties [14].

SuSy assays

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Enzyme purification and chromatography
  6. SuSy assays
  7. Determination of kinetic parameters and modelling
  8. Results
  9. Primary (Hanes–Woolf) plot analysis
  10. Product inhibition studies
  11. Modelling
  12. Discussion
  13. Acknowledgements
  14. References

Activity in the sucrose synthesis direction was measured in 100 mm Tris/HCl (pH 7.3) buffer. The assay contained 15 mm MgCl2, 0.2 mm NADH, 1 mm phosphoenolpyruvate, and appropriate concentrations of UDP-glucose and fructose. Pyruvate kinase and lactate dehydrogenase were each added to a final activity of 4 U·mL−1. NADH oxidation was monitored at 340 nm wavelength.

Activity in the sucrose breakdown direction was routinely measured in an assay containing 100 mm Tris/HCl (pH 7.0), 2 mm MgCl2, 2 mm NAD+, 1 mm pyrophosphate and appropriate concentrations of sucrose and UDP. UDP-glucose pyrophosphorylase (UDPGlcPP), phosphoglucomutase (PGM) and Leuconostoc glucose-6-phosphate dehydrogenase (G6PDH) were each added to a final activity of 4 U·mL−1. NADH production was monitored at 340 nm.

For the UDP-glucose product inhibition study, activity was measured in an assay containing 100 mm Tris/HCl (pH 7.0), 2 mm NAD+, 2 mm MgCl2 and 1 mm ATP. Hexokinase (4 U·mL−1), phosphoglucoisomerase and glucose-6-phosphate dehydrogenase were added and NADH production monitored at 340 nm.

Determination of kinetic parameters and modelling

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Enzyme purification and chromatography
  6. SuSy assays
  7. Determination of kinetic parameters and modelling
  8. Results
  9. Primary (Hanes–Woolf) plot analysis
  10. Product inhibition studies
  11. Modelling
  12. Discussion
  13. Acknowledgements
  14. References

Substrate Km values were calculated by nonlinear fit to the Michaelis–Menten equation using grafit™ version 4 for Windows™ (http://www.erithacus.com/). Initial estimates were calculated automatically by the program based on linear regression of rearranged data. Uniform weighting was used for all data points.

Kinetic parameters other than the substrate Km values were taken as the median values calculated from the experimental data. To calculate the product inhibition constants, kinetic experiments were performed at the product inhibitor and substrate concentrations as indicated in Figs 2 and 3.

image

Figure 2. UDP-glucose product inhibition. Dixon (A,C) and Cornish–Bowden plots (B,D) with sucrose (A,B) and UDP (C,D) as the variable substrates. For (A) and (B), UDP was kept constant at 0.020 mm, while for (C) and (D) sucrose was kept constant at 40 mm. 1/v, Reciprocal reaction rate; i, inhibitor concentration; s/v, substrate concentration divided by reaction rate.

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image

Figure 3. Fructose product inhibition. Dixon (A,C) and Cornish–Bowden plots (B,D) with sucrose (A,B) and UDP (C,D) as the variable substrates. For (A) and (B), UDP was kept constant at 0.020 mm, while for (C) and (D) sucrose was kept constant at 40 mm. 1/v, Reciprocal reaction rate; i, inhibitor concentration; s/v, substrate concentration divided by reaction rate.

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The program winscamp v1.2 [15] was used for kinetic modelling, using a published model of sucrose accumulation [11]. This model can be viewed and interrogated at http://jjj.biochem.sun.ac.za.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Enzyme purification and chromatography
  6. SuSy assays
  7. Determination of kinetic parameters and modelling
  8. Results
  9. Primary (Hanes–Woolf) plot analysis
  10. Product inhibition studies
  11. Modelling
  12. Discussion
  13. Acknowledgements
  14. References

The purpose of the kinetic experiments reported in this paper was to establish the reaction mechanism of sugarcane SuSy and also determine kinetic parameters needed for metabolic modelling. As far as the SuSy reaction mechanism is concerned, there are conflicting reports in the literature; some of these results do not agree with the theoretically predicted properties of the proposed reaction mechanisms (see Discussion). Hence, there was a need to establish these properties of sugarcane SuSy.

Primary (Hanes–Woolf) plot analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Enzyme purification and chromatography
  6. SuSy assays
  7. Determination of kinetic parameters and modelling
  8. Results
  9. Primary (Hanes–Woolf) plot analysis
  10. Product inhibition studies
  11. Modelling
  12. Discussion
  13. Acknowledgements
  14. References

Primary plot analysis is used to obtain information on the reaction mechanism of an enzyme; in combination with product inhibition studies, the complete mechanism can be established. Primary plots (Fig. 1) for all substrates gave straight lines with intersection points to the left of the s/v vs. s axis, which indicates a ternary complex mechanism [for a substituted (ping-pong) mechanism the intersection points are on the axis]. The substrate Ki values obtained from the intersection points of the lines are indicated in Table 1. Sugarcane SuSy exhibited Michaelis–Menten kinetics, with Hill coefficients close to 1 (data not shown), irrespective of the variable substrate, which means that sugarcane SuSy does not display cooperative binding like some other multimeric enzymes.

image

Figure 1. Primary (Hanes–Woolf) plots for the substrates of SuSy at zero initial product concentrations. (A) Sucrose at varying concentrations of UDP; (B) UDP at varying concentrations of sucrose; (C) UDP-glucose at varying concentrations of fructose; (D) fructose at varying concentrations of UDP-glucose. Lines reflect Km and Vmax values that were derived from nonlinear fit (n = 6) to the Michaelis–Menten equation as described in Materials and methods. Kinetic assays were performed as described in Materials and methods. s, Substrate concentration; s/v, substrate concentration divided by reaction rate.

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Table 1. Inhibition types and kinetic parameters for SuSyC. Parameters were determined as described in Materials and methods.; w.r.t., with respect to.
Kinetic parameter typeSubstrate
Sucrose (mm)UDP (mm)UDP-glucose (mm)Fructose (mm)
KiS2270.0860.1042.23
Km35.9 ± 2.30.00191 ± 0.000190.234 ± 0.0256.49 ± 0.61
Inhibition constantsSubstrate
UDP-glucose w.r.t. UDP (competitive)UDP-glucose w.r.t. sucrose (mixed)Fructose w.r.t. UDP (mixed)Fructose w.r.t. sucrose (mixed)
Ki0.120.184.11.8
Ki0.193.90.65

To distinguish between a random order and ordered ternary complex mechanism, it is necessary to perform product inhibition experiments, because the primary plots for these two mechanisms have the same attributes and can therefore not be used to discriminate between the two.

Product inhibition studies

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Enzyme purification and chromatography
  6. SuSy assays
  7. Determination of kinetic parameters and modelling
  8. Results
  9. Primary (Hanes–Woolf) plot analysis
  10. Product inhibition studies
  11. Modelling
  12. Discussion
  13. Acknowledgements
  14. References

Inhibition types and inhibition constants derived from Dixon and Cornish–Bowden plots for UDP-glucose (Fig. 2) and fructose product inhibition (Fig. 3) are shown in Table 1. Competitive inhibition is characterized by a series of parallel lines in the Cornish–Bowden plot, while the Dixon plot shows the lines intersecting to the left of the y-axis. Mixed inhibition shows the lines intersecting to the left of the y-axis in both plots. The inhibition patterns indicate an ordered mechanism with UDP binding first and UDP-glucose dissociating last. Product inhibition patterns for both fructose and UDP-glucose agreed fully with the predicted patterns for an ordered ternary complex mechanism [16], with UDP-glucose a competitive inhibitor with regard to UDP and a mixed inhibitor with regard to sucrose. Fructose was a mixed inhibitor with regard to both UDP and sucrose. Although only three data points were obtained for each concentration of the variable substrate, the inhibition patterns for both UDP-Glc and fructose are nonetheless clear.

The ordered ternary complex mechanism, with UDP binding first and UDP-glucose dissociating last, agrees with that proposed for Helianthus tuberosus SuSy [17] and validates the assumption made in a kinetic model of sucrose accumulation [11], although the substrate Ki values obtained experimentally differ substantially from those used in the model. The data obtained from the kinetic experiments were then incorporated in the model of sucrose accumulation, to investigate the effect of changes in SuSy kinetic parameters on the output variables.

Modelling

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Enzyme purification and chromatography
  6. SuSy assays
  7. Determination of kinetic parameters and modelling
  8. Results
  9. Primary (Hanes–Woolf) plot analysis
  10. Product inhibition studies
  11. Modelling
  12. Discussion
  13. Acknowledgements
  14. References

Kinetic parameters obtained experimentally were used to query a kinetic model of sucrose accumulation [11]. This model, constructed using the program winscamp[15], consists of 11 reactions that are either directly or indirectly involved in sucrose metabolism. Enzymes with sucrose as substrate or product are included explicitly, while others, specifically glycolysis and the enzymes phosphoglucoisomerase, phosphoglucomutase and UDP-glucose pyrophosphorylase (UGPase) are included as a single ‘drain’ reaction and a so-called ‘forcing function’, respectively. The forcing function assumes that the reactions catalysed by phosphoglucoisomerase, phosphoglucomutase and UGPase are close to equilibrium in vivo, which is supported by metabolite measurements in most tissues. The reactions are entered as rate equations in the model, which means that all the relevant kinetic parameters are needed for each enzyme. Because of the paucity of kinetic information on sugarcane enzymes most of these parameters were estimated. Enzyme levels were taken mostly from the literature on sugarcane, others were estimated. The model solves the differential equations describing the synthesis and degradation of each metabolite in order to calculate the steady-state levels. The model ‘behaves’ like a sugarcane storage parenchyma cell, in that it accumulates sucrose, with other metabolite levels fairly close to experimentally measured values.

Variable outputs from the model are shown in Fig. 4. Outputs from the original model are shown as the first bar in every panel. For all the other model variants, the equilibrium constant for the SuSy reaction was changed to 0.50 (the published model used an equilibrium constant of five in the sucrose breakdown direction [18], but this is incorrect; reported values range from 0.15 to 0.56 [19]). Also, the SuSy parameters which were input in the original model did not obey the two Haldane relationships, which relate the Keq to the Vf/Vr ratio, Km and Ki values [16]. The two equations are given below:

  • image(1)
  • image(2)

where A is UDP; B, sucrose; P, fructose; Q, UDP-glucose; and Vf and Vr refer to maximal reaction rates in the sucrose breakdown and synthesis directions, respectively.

image

Figure 4. winscamp kinetic model variable outputs. Model variants are as follows: or., original published model; corr., model with Keq and Ki values corrected (see Results); C, model with SuSyC parameters; 2*, as for C, but doubled activity; 1/2, as for C, but halved activity; 2i, model containing two SuSy isoforms, one with generic parameters, the other with experimentally determined parameters – total SuSy breakdown activity was kept the same as for the first three model variants.

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For the corrected model (Fig. 4, model variant 2) all kinetic parameters were kept the same as the values used in the published model, except the Ki value for UDP (KiA) was changed from 0.3 to 0.108 mm, and the Ki value for fructose (KiP) was changed from 4 mm to 3.92 mm in order to obey the two Haldane relationships. In order to ensure compliance with these thermodynamic relationships, the Ki values used for the models incorporating the SuSyC parameters (Fig. 4, variants 3–6) were modified somewhat from the experimental values. These modified values were (in mm), 0.103, 0.0871, 3.10 and 139 for UDP-glucose, UDP, fructose and sucrose, respectively, with Km values used in the models as shown in Table 1. Note that the modified Ki values for fructose and sucrose are both in the same range as the experimentally determined values, while the values for UDP-glucose and UDP are extremely close to the experimentally determined values.

The output variables differed appreciably between models containing two different SuSy isoforms. Sucrose, glucose, Fru-6P and UDP-glucose concentrations were all higher in model variant C than in 2. Fructose was the variable most affected by changes in the SuSy isoform in the model or changes in SuSy activity (see Discussion), although sucrose concentration also increased by about 41% in model variant C. Sucrose content was positively correlated with SuSy activity, but these changes were quite small compared with the changes in enzyme activity, at about a 4% increase and 9% decrease in sucrose for a doubling and halving of activity, respectively. Sucrose futile cycling was about 7% higher in the models containing the SuSyC isoform, compared with the model (variant 2) with the ‘generic’ SuSy. Notably, percentage conversion of hexoses to sucrose increased from 84.4 to 87.0%, and percentage carbon to glycolysis decreased from 15.6 to 13.0% in model variant C, compared with 2.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Enzyme purification and chromatography
  6. SuSy assays
  7. Determination of kinetic parameters and modelling
  8. Results
  9. Primary (Hanes–Woolf) plot analysis
  10. Product inhibition studies
  11. Modelling
  12. Discussion
  13. Acknowledgements
  14. References

It is interesting to compare the results obtained in this study with those for maize [20] and Helianthus tuberosus SuSy [17]. UDP-glucose is a competitive inhibitor with regard to UDP, and fructose a competitive inhibitor with regard to sucrose, according to both these studies. These results, however, conflict with the predicted patterns of product inhibition for an ordered ternary mechanism [16]; instead, they agree with the expected patterns for a substituted (ping-pong) mechanism. A random mechanism was proposed for SuSy from Phaseolus aureus[21], but this finding was later challenged [17]. The results of the study on sugarcane SuSy indicated that it follows an ordered ternary mechanism, with no evidence to suggest otherwise. The apparent conflict between the product inhibition patterns obtained in the studies on maize and Helianthus SuSy on the one hand and sugarcane SuSy on the other is puzzling and merits further investigation.

The kinetic data obtained in this study was used to query a model of sucrose accumulation [11]. It was found that substituting the mostly estimated kinetic parameters of SuSy in the original model with the experimentally determined parameters of the SuSyC isoform had a marked effect on most variables output by the model. The 41% increase in sucrose concentration and the more than 7 times reduction in fructose concentration were the most notable. Evidently, changes in kinetic parameters of enzymes involved in sucrose metabolism are capable of having large effects on metabolite concentrations. According to this model, expression of multiple enzyme isoforms may therefore play an important role in the regulation of metabolism, as they can be used to influence metabolite concentrations in different ways. Therefore, different SuSy isoforms may influence sugarcane sucrose levels differentially in vivo; this information can be put to use in sugarcane improvement programmes.

Changes in SuSy activity also impacted the model variables. The biggest changes were in fructose concentration, which decreased by 42% when activity was doubled, and increased by 140% when activity was halved. Incorporation of the SuSyC isoform in the model dramatically reduced the steady-state concentration of fructose compared with the model with estimated SuSy parameters, from 22.6 to 3.04 mm. This may seem alarming when compared with experimentally reported values of about 30 mm for fructose in internode five [22], but it has to be kept in mind that these experimental values assume equal distribution of fructose between the cytosol and vacuole. Up to 99% of glucose and fructose in this tissue might actually be present in the vacuole [23], and hence the low value for cytosolic fructose obtained with the modified model is not necessarily incorrect. On the other hand, one would expect the glucose and fructose values to be more or less equal, but this is not so in the modified model. Only metabolite measurement methods that can distinguish between the cytosolic and vacuolar compartments can resolve this issue.

Next, the model was expanded so that in addition to the SuSy isoform with generic kinetic parameters, it included a second SuSy isoform, with experimentally determined kinetic parameters. Total SuSy breakdown activity was kept the same as in the models with only one SuSy isoform. Modelling results with this version were very similar to the model containing only the SuSyC isoform, except for the fructose concentration, which was 67% higher. This change in the fructose concentration suggests that expressing different enzyme isoforms simultaneously may add to the regulatory capabilities that plants have over their metabolism, in addition to expressing isoforms in spatially and temporally separate ways.

Reducing SuSy activity 10-fold results in the fructose concentration increasing about 17-fold and halving of sucrose concentration (data not shown). This is consistent with experimental data that show that SuSy participates in sucrose synthesis in younger internodes [24]. It would be insightful to modify the model for a mature internode, and then see what effects changes in SuSy activity have. It would be best to establish enzyme activity levels for all the enzymes incorporated in the model simultaneously with a single enzyme extract, in order to avoid the fragmented and approximate data set used for the current model.

The utility of modelling sucrose metabolism was illustrated in this work; the results obtained could not easily have been predicted by other means. Computational systems biology approaches can therefore play a very useful role in studying processes that impact on sucrose accumulation, such as futile cycling. Futile cycling is an energetically wasteful process, as for sucrose to be resynthesized the hexoses have to be phosphorylated again at the expense of ATP, and therefore reduction of this process in sucrose accumulating tissue is an important goal. The modelling results indicate that, at least in a fairly young internode, sucrose futile cycling is not greatly affected by specific SuSy isoforms. This may not be the case in a mature internode; therefore mature tissue should also be modelled in order to answer this question.

In conclusion, kinetic modelling can be used not only to predict the effects of variation in the activity or kinetic parameters of enzymes catalysing different reactions, but can also yield information about the metabolic effects of the presence of more than one isoenzyme, such as SuSy isoforms in sugarcane. This makes possible much more informed decisions on manipulation strategies for yield improvement in any system that can be modelled this way. Obtaining the reaction mechanisms and kinetic parameters of all enzymes involved in such a system is an essential step in this approach.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Enzyme purification and chromatography
  6. SuSy assays
  7. Determination of kinetic parameters and modelling
  8. Results
  9. Primary (Hanes–Woolf) plot analysis
  10. Product inhibition studies
  11. Modelling
  12. Discussion
  13. Acknowledgements
  14. References
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