To what extent has genetic manipulation of the Calvin cycle forced the reappraisal of our understanding of the control of metabolic pathways in plants. What do studies of transgenic plants reveal about the integration of metabolism?

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Chris Holland        Jesus College

To what extent has genetic manipulation of the Calvin cycle forced the reappraisal of our understanding of the control of metabolic pathways in plants. What do studies of transgenic plants reveal about the integration of metabolism?

Within the past decade advances in genetics and molecular biology has facilitated brand new ways of looking at metabolic processes. Far from the traditional reductionalist approaches of the previous years, we are able undertake a more holistic approach towards understanding metabolic pathways and networks. The most important advance has been a move away from inferred models based on in vitro characteristics of enzymes to real-time studies in vivo of enzymes at work. One of the best understood metabolic networks (and also among the best funded in plants) is the primary pathway for Carbon fixation, the Calvin cycle. In this essay I shall describe how the regulation of metabolic pathways was originally approached, how the use of genetics has changed this approach and describe in detail some experiments on enzymes in the Calvin cycle and how the results from these has caused a reappraisal of our understanding of how metabolism is controlled in plants.

Traditional Methods

Metabolic pathways consist of a series of chemical modifications to a compound which results in substrates being turned into products. At each step of the way enzymes are used in order to allow the reaction to occur at physiological temperatures and at a speed conducive to homeostasis. The regulation of the flux of a pathway has been one of the key questions in understanding metabolism; is flux regulated by a series of steps that act as a bottleneck to the system or by co-limitation by several enzymes.

Generally speaking enzyme activity can be modified via two different mechanisms. For short-term, “fine” changes, due to a change in the environment, enzymes can be modified by altering the existing enzymes kinetics, changing levels of substrate, inhibitors or activators and by post-translational modification (e.g. carbamylation of rubisco).  Longer-term “coarse” changes, such as those incurred during development, require the amounts of enzymes to be altered, through modification of the transcription of the gene or by protein turnover.

Original thinking behind regulation of flux in a pathway has been that enzymes catalysing steps far from thermodynamic equilibrium (i.e. irreversible) are best suited for regulation as they provide a bottleneck for the pathway that relies on their catalytic abilities. Enzymes that catalyse steps that are at or near thermodynamic equilibrium (i.e. readily reversible) would be poor sites for regulation as they are likely to be present in high numbers and control will be unable to favour either the products or substrates in the reaction. Evidence to support this view comes in the form that irreversible enzymes tend to have regulatory properties, including allosteric regulation and/or post-translational regulation. Also a good candidate for a regulatory enzyme would show some reciprocal relationship between its concentration of substrate and overall flux of the pathway. For example if the in vivo concentration of an enzymes substrate is reduced and subsequently the flux of the pathway it is involved in also goes down then it can be hypothesised that the enzyme catalysing that step is responsible in part for the overall flux. Given these hypothetical qualities Newsholme and Start in 1973 developed a theoretical analysis for the study of regulation. Stating that an enzyme is likely to regulate the flux of a pathway if it fulfils these criteria:

  1. They catalyse an irreversible reaction.
  2. They possess regulatory properties.
  3. They show characteristic reciprocal relationship between the in vivo flux and their substrate concentration.

Using this framework enzymes were identified, purified and studied for their kinetic and regulative properties in vitro and by correlation with in vivo expression patterns, changes in fluxes and so on, it was then possible to frame a hypothesis about how the enzymes was regulated. This approach produced a large amount of information regarding individual properties of enzymes but there are many problems associated with this approach which made it very difficult to conclusively prove the models created. It is not to say that all study went on in vitro just that there was a large imbalance between the two due to the lack of technical ability to study in vivo systems properly.

The fundamental flaw in this analysis is that enzymes are integral to the pathway and once an enzyme is removed from its native surroundings it behaves differently. Any hypothesis being drawn from it can be misleading, much like observing an animal’s behaviour in the zoo and inferring how it reacts in the wild. By limiting analysis to only enzymes that facilitate irreversible reactions resulted in many potential (although thought unlikely) site for regulation were missed out. Also showing a correlation between flux and enzyme activity in vitro does not imply that the enzyme is primarily responsible for that change and merely because they have regulatory properties does not mean that they are used in vivo.

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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” ...

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