Allosteric Effectors

4.4. Sucrose Phosphate phosphatase

In general, SPS and sucrose-P phosphatase appear together in tissues, but the activity of sucrose-P phosphatase exceeds the maximum activity of SPS by about 10-fold (107). Although this suggests that phosphatase may not be limiting for sucrose formation, it is likely that the enzyme is not operating at maximum velocity in situ. SPS is able to catalyze a rapid flux in the direction of sucrose synthesis if its products are effectively removed. The enzyme has been partially purified from sugarcane, and has a slightly acidic pH optimum, is specific for sucrose-P as substrate, and requires Mg2+ for activity (108). The enzyme consisting of two similar subunits (55 KDa) is sensitive to sucrose. This property could act as an important mechanism to limit the accumulation of sucrose (69).

4.5. Pyrophosphate: fru-6-P 1-phosphotransferase and PPi metabolism

Most plant tissues contain substantial activities of an enzyme, termed, pyrophosphate:fructose-6-P 1-phosphotransferase (PFP), which catalyses a reversible phosphorylation of fru-6-P using PPi (109). PFP is a cytosolic enzyme and is activated by fru-2,6-P2 (110). Since the enzyme catalyzes a reversible reaction, it could either utilize PPi as an energy source during glycolysis or generate PPi by catalyzing the reverse reaction in which fru-1,6-P2 and Pi are converted to fru-6-P and PPi (69). The enzyme purified from a large number of plant sources is a tetramer with molecular mass of about 250 KDa (111) containing two different kinds of subunits, termed a and p . PPi is a powerful inhibitor of the reverse (fru-6-P forming) reaction of PFP, inhibiting competitively with fru-l,6-P2 (112) with Ki value of 10-15 nM. Pi, on the other hand, inhibits forward reaction non-competitively. However, reports for mixed and competitive inhibition by Pi are also available (49). Fru-2,6-P2 acts as an activator of the enzyme and activates the forward reaction by increasing the Vmax and lowering the Km (fru-6-P), often by a factor of 10 or more (112). The effect on the Km (PPi) is smaller and more varied with reports of a decrease, no effect, or a small increase. Fru-2,6-P2 activates the reverse reaction (fru-6-P forming) by decreasing the Km (fru-1,6-P2) more than 10 fold. The affinity for fru-2,6-P2 is increased by fru-6-P and fru-l,6-P2, decreased by Pi and by many phosphorylated intermediates, organic anions, and inorganic anions (49).

Fig. 4. Schematic representation of the regulation of leaf SPS by reversible seiyl phosphorylation. An increase in glu6P and a decrease in Pi, would favour dephosphorylation /activation of SPS. Another important factor may be light modulation of the regulatory properties of SPS-PP, and changes in cytosolic [Ca2+],

One molecule of PPi is formed in the reaction catalyzed by UDPGPPase for every molecule of sucrose that is synthesized, so PPi must be hydrolyzed at rates of 10-15 nmol/mg chl/hr during rapid photosynthesis. PPi thus formed is hydrolyzed via a cycle between PFP and the cytosolic FBPase, in which PFP converts PPi and fru-6-P to fru-l,6-P2 and Pi, and the fru-l,6-P2 is then hydrolyzed by the enzyme FBPase. The activity of this cycle would depend on the level of fru-2,6-P2> because of the differential effects of fru-2,6-P2 on the forward and reverse reactions of PFP. Lower levels of fru-2,6-P2 favour removal of PPi, but increasing fru-2,6-P2 activates the reverse (PPi-generating) reaction and reduces the rate at which PPi is removed. Simultaneous changes of the metabolites could amplify this response, because they influence the rate of the reverse reaction, as well as its sensitivity to activation by fru-2,6-P2. For example, during a feedback inhibition of sucrose synthesis, higher fru-2,6-P2 restricts the cytosolic FBPase and leads to an accumulation of fru-l,6-P2, while Pi may decline. Higher fru-1,6-P2 and lower Pi directly increase the rate of reverse reaction, and also increase its sensitivity to activation by fru-2,6-P2 (113, 114).

4.6. Regulation of fru-2,6-P2 concentration:

Fru-2,6-P2 is a regulatory metabolite, whose turnover is altered in response to a variety of signals about metabolic conditions in the leaf, allowing these signals to be amplified and integrated as a change in the concentration of fru-2,6-P2 (69). Fru-2,6-P2 then interacts with the target enzymes to readjust the fluxes and metabolite pools in the cytosol. The level normally lies between 80-500 pmol/mg chl, which would be equivalent to a total concentration of 4-25 nM, if this fru-2,6-P2 were all free in the cytosol (volume 20 pl/mg chl). The enzymes synthesizing and degrading fru-2,6-P2 as well as cytosolic FBPase are mainly and entirely located in the mesophyll cells. Fru-6-P2-kinase and fru-2,6-P2ase catalyze the synthesis and degradation of fru-2,6-P2, respectively (49). Fru-6-P and Pi activate the enzyme fru-6-P 2-kinase and inhibit fru-2,6-P2ase. The enzyme fru-6-P 2-kinase is inhibited by PGA and DHAP at concentrations under 1 mM. These concentrations are similar to those found in the cytosol (49, 69). Increasing fru-6-P relieves the inhibition, but shows a complex interaction with Pi. PGA inhibits strongly in the absence of Pi. This inhibition decreases with increase in the concentration of Pi. Interaction of these four metabolites with fru-6-P 2-kinase and fru-2,6-P2ase allows a varied response of the fru-2,6-P2 concentration to different metabolic conditions. Though fru-6-P,2-kinase and fru-2,6-P2ase from animal tissues are regulated by phosphorylation via a cAMP-dependent protein kinase, which in turn is regulated hormonally, there is no conclusive evidence of similar regulation of these enzymes in plants. There is, however, evidence that plant fru-6-P,2-kinase is regulated covalently in addition to its regulation by metabolites (48).

As the rate of photosynthesis increases, there is a decrease in the level of fru-2,6-P2. This is due, at least in part, to regulation of fru-6-P 2-kinase by three carbon effectors like PGA and DHAP. In general, an increase in the rate of photosynthesis will be accompanied by an increase in the concentrations of these C3 effectors, which will inhibit fru-6-P 2-kinase and lead to a decrease of fru-2,6-P2 (49).

4.7. Co-ordinated control of sucrose synthesis by SPS and cytosolic FBPase

Although the cytosolic FBPase and SPS are the regulatory enzymes in the pathway of sucrose biosynthesis, it is important to realize that regulation is the property of an entire pathway rather than of individual processes. For an individual enzyme to contribute significantly, it must possess properties that allow its activity to be controlled, but the impact of the enzyme on the flux depends on its integration into the whole pathway. When the flux through a pathway is modified by the activation of one enzyme, this will affect the concentration of both substrate and products of this enzyme. Changes in these metabolites may then affect the activities of other enzymes in the pathway, whose activity must be changed if the flux through the entire pathway is to be modified. This suggests that the regulation of a given pathway requires interaction and co-ordination between different enzymes. We discuss below how metabolic 'fine' control and 'coarse' control of the cytosolic FBPase and SPS may be co-ordinated to enable sucrose synthesis to respond to alterations in the availability of photosynthate or the demand for sucrose.

Concentration of triose-P increases in response to increased rates of photosynthesis, and in turn, the concentration of fru-2,6-P2 decreases two-to three fold. As a result, the activity of the cytosolic FBPase rises, and more fru-6-P is formed. Since fru-6-P and glu-6-P are in equilibrium via phosphogluco isomerase, increased production of fru-6-P will result in a greater glu-6-P concentration in the cytosol and consequently SPS will be activated. Thus it is evident that increased rates of photosynthesis will lead to increased rates of sucrose formation as a result of co-ordination of the regulatory properties of the cytosolic FBPase and SPS (Fig. 5). These two enzymes remain inactive until a threshhold concentration of triose-P or hexose-P is attained, respectively. They will then be strongly activated by further small increase of these metabolite pools. SPS is also subject to 'coarse' control during light-dark transitions in many plants, leading to a change in its substrate affinity. Such 'coarse' changes will interact with the fine metabolite control and allow SPS to be activated by small changes of the hexose-P pool, or even without any changes.

During periods when sucrose is produced more rapidly than it can be exported, sucrose accumulates within the leaf. As sucrose accumulates, the concentration of fru-2,6-P2 increases two to three fold. This increase in fru-2,6-P2 would decrease the activity of the cytosolic FBPase and result in decreased rates of sucrose synthesis. The mechanism(s) whereby fru-2,6-P2 concentration increases as sucrose accumulates within the leaf is not completely understood. However, it appears that 'coarse' control as well as metabolic 'fine' control are involved, and there may be species differences (49, 69). Thus, it appears that coarse control of enzyme activity is part of a feedback mechanism that co-ordinately controls SPS and the cytosolic FBPase while alterations of the cytosolic fru-6-P may operate as a fine control that co-ordinates the response to a 'coarse' control at two different sites (69). Together these mechanisms restrict the rate of sucrose formation and lead to an accumulation of metabolites in the cytosol. The decreased supply of Pi then stimulates starch synthesis inside the chloroplasts. This is how a leaf alters the partitioning of carbon between sucrose and starch when demand for sucrose is less than its supply.

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