A New Approach
A step forward in thinking about how metabolic pathways should be analysed was the differentiation between enzymes “regulatability” and their “regulatory capacity” (Hoffmeyer and Cornish-Bowden1991). In order to address the problems described above, Hoffmeyer made the distinction between an enzymes potential to be regulated in vivo (regulatability), and the contribution that enzymes regulation makes to the overall flux of the system (regulatory capacity). The theoretical analysis traditionally applied to metabolism only identified enzymes with a high “regulatability” it does not provide us with a set of logical criteria with which to understand an enzymes “regulatory capacity” or help define exactly which enzymes are key control points in the system.
In order to assess the “regulatory capacity” of an enzyme, small changes in the activity of an enzyme must be made in vivo and measuring the effect it has on the flux through the whole pathway. This can be visualised by plotting the pathway flux against the enzyme activity normalised to the wild type value.
If the enzyme is the sole control point, limiting the entire flux of the system, then there is a linear and strictly proportional relationship between enzyme activity and flux of the system. When the enzyme together with other enzymes, co-limits flux there will be a curvilinear response with a finite but non proportional slope in the range corresponding to wild type plants. Where the enzyme has no significant control in the system the slope will be zero in the wild type range. What is important to note is that at some point the flux will change according to enzyme activity although this only shows that the enzyme is essential or redundant not whether it plays a role in regulation of flux.
The degree to which an enzyme is said to control the flux in a pathway is referred to as the enzymes flux control coefficient. Derived by Kacser and colleagues from the above graph and formalised into this equation.
Where J is the flux and E is the enzyme activity normalised to wild type and C is the flux control coefficient. Since this relates to the gradient of said graph it is deducible that C will always take a value between 0 (non-limiting) and +1 (strictly limiting). For the entire pathway the sum of all enzymes flux control coefficient must equal 1 (in the wild type plant) otherwise there are factors that play a regulatory role.
The value of an enzyme’s flux control coefficient cannot be predicted from its properties, or the thermodynamic state of its reaction. The model developed by Kacser predicts that the values of flux control coefficients of the enzymes in the pathway emerge from an interaction between all the enzymes of the pathway. Due to this value depending on the interactions within the system it can only be determined by a holistic approach. Experimental measurements of flux control coefficients started with estimates from metabolic models, using inhibitors to titrate out the enzyme activity in vivo and careful control of substrate concentrations. The problem with these assumptions is that they require a simplification of the system based on prior knowledge of the enzymes and their kinetic properties and also that specific inhibitors and substrates have been identified and can be modulated in vivo.
Another approach is to search for mutants that express varying levels of the enzyme in question and assess its flux with comparison to a wild type. Problems arise here due to the fact that the enzyme in question must confer some sort of selective phenotype for screening processes and the majority of metabolic processes either have no easily identifiable phenotype or it is covered up by redundancy in the system. In order to accurately obtain the flux control coefficient a range of mutants must be identified each displaying different levels of enzyme activity, you cannot plot a graph with two points. With this in mind it appears that mutant screening for different enzyme expression levels is a hopeless task although some groups have managed to achieve it.
A Solution?
With the advent of molecular biology and an increased understanding of genetics, the last decade or so has provided a methodology that has revolutionised the way in which the study of flux control coefficients has been approached. Now it is possible to directly manipulate levels of individual enzyme activity by altering their expression patterns though either upregulation (as in ectopic expression) or downregulation (antisense, cosupression (is it really understood yet?) and targeting of foreign proteins (I don’t understand the point of them, it is in order to move orthologues to the right place as in Miyagawa)) of the genes that encode them. Another decisive advantage of transgenic plants it that they reflect the importance of the enzyme not only acting in the pathway but for whole plant development.
The most popular method of creating mutants with varying levels of expression is to use antisense technology and rely on random insertion and position effects to generate different enzyme activities (is this RNAi now?). Others have been used and have shown promising results especially when orthologues of native genes are ectopically expressed which do not undergo normal regulation (Miyagawa 2001). Ideally plants with an activity of enzyme relative to wild type ranging from 60-70% right down to 10% above these values the enzyme activity is hard to determine exactly due to compensatory effects and below the enzymes do not show regulation but necessity.
The Calvin Cycle
One of the fields to have benefited the most from this research is the primary carbon fixation cycle or Calvin cycle. Photosynthetic carbon metabolism in higher plants is thought to be one determining factor in plant growth and yield. The cycle combines a five carbon compound (ribulose-1,5-bisphosphate) with a molecule of CO2 and through a process of reduction creates two triose phosphates which can be siphoned off into other metabolic pathways or used to regenerate the ribulose 1,5-bisphosphate. Overall there is a net gain in carbon, hence the term carbon fixation cycle.
This is a diagram of the Calvin cycle showing the pathway of carbon as it is fixated. Of the 13 reactions involved in the cycle 11 of them are catalysed by enzymes.
Irreversible reactions are catalysed by the following 4 enzymes.
- ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco)
- sedoheptulose-1,7-bisphosphatase (SBPase)
- fructose-1,6-bisphosphatase (FBPase)
- ribulose-5-phosphate kinase (PRK)
Although there are many flaws with the traditional approach these enzymes had already been well characterised and their encoding sequences were well known, making them perfect candidates for investigating the contribution they make to flux in the Calvin cycle.
One question to be asked is why regulate the Calvin cycle at all? The surrounding environment is not static and the system must be able to make changes in different light intensities and CO2 concentrations. Since the Calvin cycle relies upon the products of the light dependant reactions in photosynthesis ATP/NADPH, it must be able to switch off during the dark otherwise it uses carbohydrates in order to create the ATP/NADPH which in turn is used to make new carbohydrates. Such changes in the relative concentrations of intermediates will effect other pathways which share them, such as glycolysis and oxidative PPP.
I shall now outline some experiments performed on these enzymes and in turn describe what effects the results have had on the traditional ideas regarding regulation of the Calvin cycle.
Ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco)
Rubisco is the most abundant enzyme on the planet. It is responsible for the fixation of CO2 in the Calvin cycle and was generally thought to regulate (limit) the rate of photosynthesis. It comprises of approximately 40% of the total protein in a leaf and represents a key site in the carbon and nitrogen economy of the plant. Rubisco was well known to be a highly regulated enzyme, undergoing carbamylation in order to become active which then allows a host of activators and phosphorylated intermediates to interact with it. What was not known was its “regulation capacity”. Stitt and Schulze (1994) used a set of “antisense” tobacco plants in order to determine the flux control coefficient. Under moderate light, ambient photosynthesis was only slightly inhibited when rubisco was reduced to about 60% of the wild type variants and a control coefficient of 0.05-0.15 was estimated.
This had massive implications for the traditional thinking. Here was an enzyme that was highly regulated yet appeared to contribute very little to the overall flux of the system. It was thought that since the rate of photosynthesis stayed nearly the same in the mutant 60% plants they must be using rubisco in a more efficient manner than their wild type counterparts. Studies showed that although there was a slight increase in substrates to compensate for the loss that would only account for 15% of the “missing” protein. What had occurred was that the level of the carbamylated (active) enzyme was increased in order to compensate. It was clear that redundant mechanisms were present in order to adjust the system to perturbations, revealing a new level of control and feedback within metabolism.
Perhaps one of the most interesting results to emerge from the study was that the flux control coefficient varied according to the conditions photosynthesis was measured in. It was already known that in moderate light C 0.05-0.15 but when plants were grown in low light then suddenly plunged into bright light (altering the rate of photosynthesis) there was a near proportional relationship between the amount of Rubisco and the rate of photosynthesis (C >0.9!). This result was echoed when repeated with low CO2 growth levels and yet the converse was obtained when measurements were taken in 5% CO2. This clearly shows that an enzymes regulatory capacity depends on the short term conditions under which the flux was measured. And also due to the long term conditions the plant was subjected to before the change in conditions and flux was measured. (There doesn’t seem to be any difference between 4.1 and 4.2 in terms of experiments in Stitts paper the first will be the last)
In summary the analysis of Rubisco mutant revealed that it plays a much smaller part in regulation of the Calvin cycle, thus control must be shared with other enzymes. The only case where rubisco appears to be the major limitation in the Calvin cycle is in conditions of high irradiance and temperature. There exists a certain amount of redundancy within the system which can compensate for changes in the levels of enzyme expression. Also the enzymes regulatory capacity depends on the conditions it is measured in and the conditions it was produced in (short term and long term effects).
Sedoheptulose-1,7-bisphosphatase (SBPase)
Work by Harrison et al (1998) on SBPase was important to the elucidation of the Calvin cycle regulatory components as it catalyses a reaction shortly after the branching of the cycle and was thought to be important in carbon portioning to sucrose and starch. In vitro studies of the enzyme showed that it is regulated by a number of different factors in particular thioredoxin f, Mg2+ and finer regulation is achieved by its products. No natural mutants have been reported showing differing levels of SBPase activity and so Harrison used basic antisense inhibition of the gene in order to create a series of mutants with different expression levels.
They created a series of mutants with enzyme activities ranging from 71%-7% wild type without any detectable changes in other Calvin cycle enzymes. However a reduction to just 57% of the wild type resulted in an impairment of photosynthesis which when compared to other enzymes in the network which require reductions between 65-90% before an effect is seen implies that SBPase has a much greater effect on overall flux in the pathway. However it does not appear that a C value was calculated. Because no other enzymes in the cycle were altered in terms of expression it implies that the redundancy mechanisms for a reduction in SBPase activity are present elsewhere in the metabolic network. Studies of carbohydrates show a shift in partitioning towards soluble carbohydrate.
What was learnt from this was that SBPase was not in as high concentrations in the plant as originally thought and that the redundancy mechanisms did not compensate for that specific pathway but altered the resource partitioning of the plant. This could be because since the enzyme was involved in a branching of the pathway it was a more efficient way of adapting.
Fructose-1,6-bisphosphatase (FBPase)
Much the experiment as above was carried out by Kossmann et al (1994) with FBPase and found C to be much lower than predicted. One interesting experiment was performed by Miyagawa Tamoi and Shigeoka (2001). By ectopically expressing a cyanobacterial FBPase/SBPase (a unique multifunction enzyme) targeted to chloroplasts in transgenic tobacco plants, they were able to obtain increased levels of photosynthesis at atmospheric conditions. Other attempts at ectopic expression using genes from the same or closely related species has resulted in problems regarding co-suppression. By using an orthologue to these enzymes which used amino acid codons not common to the plant it was able to escape the normal constraints of gene silencing and regulation. Thus the enzyme was truly removed from the loop of regulation in the Calvin cycle and the effects could be noted. The results showed that increased levels of FBPase/SBPase activity increased the level of photosynthesis, implying that both FBPase and SBPase are responsible in part for flux in the pathway.
Ribulose-5-phosphate kinase (PRK)
Paul et al (1995) investigated PRK activity using transgenic tobacco plants which produced results truly contrary to the traditional predictions. In normal growth conditions the flux control coefficient of PRK was found to be at or very near zero. Levels in enzyme could be reduced 85% before carbon assimilation was effected. It was found that in these conditions PRK is in a large excess with only 6% maximal PRK activity required to maintain CO2 rates observed in the wild type. Thus regulation of the fine feedback can compensate for large changes in gene expression.
The search for other candidates
So far the initial candidates for controlling flux in the Calvin cycle had yielded little information about the major components involved. The flux control coefficients for the aforementioned enzymes hardly add up to 1 in normal growth conditions. Focus moved from examining the enzymes that catalysed irreversible reactions to those that catalyse reversible (non regulated) ones and to everyone’s surprise it appears that they are responsible for the majority of flux control in the Calvin cycle.
Plastid Aldolase
A 30% decrease in aldolase activity results in a small but significant (5-10%) inhibition of ambient photosynthesis., and a drop below 30% resulted in a severe inhibition in plants growing under normal conditions (Haake et al 1998). Further tests were performed to support this by Stitt (1999) and resulted in the following conclusions. The results strongly indicate that decreased expression of aldolase inhibits photosynthesis for different reasons in low and high light: in low light photosynthesis is inhibited due to a restriction in the regeneration of RuBP as expected because aldolase catalyses two reaction in the regenerative part of the Calvin cycle, whereas in high light photosynthesis is inhibited due to an inhibition of starch synthesis, the resulting accumulation of phosphorylated intermediates and depletion of the free inorganic phosphate. This is the first example of how an enzyme can regulate flux through two different mechanisms depending on the conditions.
Transketolase
A final example of how metabolic networks interact with one another in terms of flux control can be seen with transgenic tobacco plants expressing antisense transketolase (Henkes et al 2001). Transketolase (TK) catalyses reactions in the Calvin cycle as well as in the oxidative pentose phosphate pathway (OPPP) and produces erythose-4-phosphate, which is a precursor for the shikimate pathway leading to phenylpropanoid metabolism. Mutants were created and the results were as follows. A 20-40% reduction of TK activity inhibited ribulose-1,5-bisphosphate regeneration and photosynthesis. The inhibition of photosynthesis became greater as irradiance increased across the range of growth conditions. TK almost completely limited the maximum rate of photosynthesis in saturating light and saturating CO2. Further inhibition of enzyme activity resulted in decreased levels of the aromatic amino acids and the major phenylpropanoids. Here we see the flux of one pathway directly affecting the flux of another, showing a true network of metabolism. The simplest explanation for this observation is that erythose-4-phosphate has been decreased which limits the flux into the shikimate pathway.
Discussion
Throughout this essay I have broached different observations that have required plant physiologists to reassess their initial preconceptions regarding the Calvin cycle. Here is a brief summary of the point observed.
- The enzymes involved in metabolic pathways are substitute to much regulation by many different factors, but this does not necessarily mean they have a large “regulatory capacity”.
- The overall control of flux in normal conditions is controlled by a number of enzymes each playing a small part. Control is not solely reserved for enzymes that catalyse irreversible reactions but evidence is mounting to show that reversible enzymes are involved as well.
- The contribution of a given enzyme to the control of flux is dependent on both the conditions flux is measured in and the conditions in which the plant has accumulated to.
- In general adjustments in “fine” regulation are usually enough to compensate for changes in gene expression. However redundancy mechanisms are in place in case fine regulation is unable to cope.
- Contrary to traditional thinking “non regulated” enzymes are not present in excess, usually only 2 to 3 fold over the minimum required to avoid severe flux limitations.
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“Regulated” enzymes are often present in excess, yet only reach a small percentage of their maximum activity (how did they get so good in the first place if not selected for?). This would provide a redundancy zone to allow decreased gene expression to be accounted for.
The use of transgenic plants provides us with a powerful tool but does not provide us with all the answers. There are many limitations in using transgenic plants which I do not have time to talk about here, but sufficed to say that we are not all the way home when it comes to understanding metabolic systems.
Bibliography
Stitt, M. and Sonnewald, U. (1995) Regulation of metabolism in transgenic plants. Ann. Rev. Plant Phvsiol. Plant Mol. Biol. 46: 341-368.
Stitt, M. & Schulze, D. (1994) Does Rubisco control the rate of photosynthesis and plant
growth? An exercise in molecular ecophysiology. Plant, Cell and Environment 17, 465-487. (Read only so far as it appears biochemical after a while this review strays into plant physiology, plant growth, and ultimately the bucket-and-spade end of botany)
Stitt, M. (1995) The use of transgenic plants to study the regulation of plant carbohydrate
metabolism. Aust. J. Plant Physiol. 22. 635-646.
Stitt, M. (1999) The first will be last and the last will be first: non-regulated enzymes call the tune? In Plant Carbohydrate Biochemistry, Bryant, J.A., Burrell, M.M. & Kruger, N.J., eds. Bios Scientific, Oxford pp. 1-16. (ask NJK for pdf file)
Miyagawa, Y., Tamoi, M. & Shigeoka, S. (2001) Overexpresion of a cyanobacterial fructose
l,6-sedoheptulose-l,7-bisphosphatase in tobacco enhances photosynthesis and growth. Nature Biotechnol. \ 9 965-969.
Harrison EP, Willingham NM, Lloyd JC, Raines CA (1998) Reduced sedoheptulose 1,7-bisphosphaase levels in transgenic tobacco lead to decreased photosynthetic capacity and altered carbohydrate accumulation. Planta 204: 27-36.
Haake, V., Zrenner, R., Sonnewald, U. and Stitt, M. (1998) A moderate decrease of plastid
aldolase activity inhibits photosynthesis, alters the levels of sugars and starch, and
inhibits growth of potato plants. Plant, L 14: 147-157.
Henker, S., Sonnewald, U., Badur, R., Flachmann, R. and Stitt, M. (2001) A small decrease of plastid transketolase activity in antisense tobacco transformants has dramatic effects on photosynthesis and phenylpropanoid metabolism. Plant Cell_\3: 535-551.More Specific References (very detailed, but demonstrating various points!)
Paul, M.J. et al. (1995) Reduction in phosphoribulokinase activity by antisense RNA in transgenic tobacco: effect on CO2 assimilation and growth at low irradiance. Plant J. 7: 535-542.