Propagation of Metabolic Perturbations through Networks

The metabolic consequences of altering the amount of an enzyme are unlikely to be confined to a single pathway. A clear illustration of the extent of the interactions that occur between pathways is provided by a study of transgenic tobacco lines in which the amount of transketolase was selectively decreased (Henkes et al., 2001). These lines displayed a near-proportional decrease in the maximum rate of photosynthesis in saturating CO₂ and a smaller inhibition of photosynthesis under normal growth conditions. This inhibition was accompanied by large decreases in the steady-state levels of RuBP and 3PGA, smaller decreases in the amounts of triose phosphates and fructose 1,6-bisphosphate, and a large increase in the amount of fructose 6-phosphate. These changes are entirely consistent with restrictions in the two reactions of the Calvin cycle catalyzed by transketolase and suggest that the immediate cause for the decrease in photosynthesis is a restriction in the ability to regenerate RuBP (Fig. 1.3). Thus, the effect of reduced transketolase appears to be similar to that obtained when the aldolase content was decreased under low light (Fig. 1.2A). However, in contrast to the consequences of manipulating aldolase content, a decrease in transketolase also caused a disproportionately large decrease in the levels of aromatic amino acids, intermediates of the phenylpropanoid pathway, and secondary products such as chlorogenic acid and lignin. These observations suggest that the level of transketolase has a major impact on the channeling of intermediates into the shikimic acid pathway and the likely explanation for this effect is that the metabolic network responds to a decrease in the amount of transketolase by decreasing the amount of erythrose 4-phosphate (Fig. 1.3). Consequently, flux into the shikimic acid pathway is restricted by the supply of erythrose 4-phosphate and phenylpropanoid metabolism is constrained by the corresponding decreased provision of aromatic amino acids.

The multiple responses to reducing transketolase highlight the extent of integration within the central metabolic pathways and the potential difficulties in attempting to modify flux through a specific section of the metabolic network. In particular, the results suggest that major changes in secondary metabolism may require appropriate reprograming of primary pathways to ensure an adequate supply of the necessary precursors. Corroborative evidence that the formation of secondary products may be limited by the availability of primary precursors is provided by a report that a decrease in the levels of aromatic amino acids due to ectopic expression of tryptophan decarboxylase led to decreases in the amounts of chlorogenic acid and lignin in transgenic potato plants (Yao et al., 1995).

FIGURE 1.3 Effect of decreased transketolase content on photosynthetic intermediates in tobacco plants (Henkes et al., 2001). Changes in the steady-state levels of Calvin cycle intermediates in transketolase-antisense lines are compared with those in wild-type plants grown under the same conditions. The reactions catalyzed by transketolase are indicated by dotted lines. Symbols refer to the following changes inmetabolite content: ↑, increase; ↓, decrease.

In fact both the structure and chemical organization of metabolic networks suggest that transketolase is unlikely to be unique in the manner in which changes in its activity influence other metabolic processes. This view is supported by a theoretical analysis of the potential metabolic interactions for each of the intermediates of glycolysis and the oxidative pentose phosphate pathway (Table 1.1). Although there is considerable variation between compounds, on average each metabolite is a reactant for about 20 enzymes, and either activates or inhibits a further 22 enzymes. These values provide only a crude estimate of the complexity that arises through the multiplicity of ligand-binding interactions and the estimate is in any case very dependent on the extent to which all potential inhibitory and stimulatory responses have been identified for the selected enzymes. Even so, the analysis suggests that perturbation of the level of any metabolite within the central pathways of carbohydrate oxidation has a very strong likelihood of affecting several other reactions, thus allowing the consequences of the initial change to propagate widely throughout the network. Such considerations further emphasize the integrated nature of the central metabolic pathways and the difficulties that are likely to be encountered in attempting to modify individual processes selectively.