Flux Control Analysis

Metabolic flux in a pathway is the consequence of the reactions of the enzymes involved in the pathway under a given condition, including changes in the concentration of metabolites. Generally, the contribution of any individual enzyme to the whole metabolic flux varies considerably, that is, while flux control is distributed over the entire pathway, enzymes in the pathway carry different weight. Often, the flux-limiting step is located at the first metabolic step of either a pathway or branch point and at those steps with a large free energy change that are virtually irreversible. However, the contribution to metabolic flux of an enzyme catalyzing a reversible reaction may also be high, when the catalytic efficiency of the enzyme (k_cat) and/or expression or steady-state amount (k_m) of an enzyme are low.

Antisense technology has provided an opportunity for precise analysis of flux control in metabolism (Stitt and Sonnewald, 1995). Metabolic flux analysis is a tool whereby metabolic flux in a system is quantified. The flux control coefficient (C^J_E = ΔJ/ΔE) is the mathematical expression of the effect of a change in the relative amount of enzyme ΔE (generally corresponding to the enzyme activity) on the metabolic flux (J) (Kacser, 1987; Stephanopoulos et al., 1998). An enzyme with C^J_E closer to zero contributes little to the flux and an enzyme with C^J_E closer to 1 contributes more significantly.

The PCR cycle includes 13 reactions catalyzed by 11 enzymes (Robinson and Walker, 1981). The effect of changes in the amount of these enzymes has been analyzed by downregulating the genes coding for the enzymes. Photosynthesis was not affected by decreasing the amount of RuBisCO at low light intensities over a large range of reduction but eventually its amount became limiting (Krapp et al., 1994; Quick et al., 1991). According to flux criteria, the C^J_E value of RuBisCO was near unity at saturating light intensities in tobacco and rice transgenic plants (Makino et al., 1997; Masle et al., 1993). Decreasing the enzyme level of glyceraldehyde 3-phosphate dehydrogenase in transgenic tobacco then caused the concentration of RuBP to decrease, but photosynthetic CO₂ fixation was not affected until the RuBP level had decreased to less than half the wild-type level (Price et al., 1995). A reduction in fructose 1,6-bisphosphatase (FBPase) amount to below 36% of wild type lowered the rate of photosynthesis (Koßmann et al., 1994). The C^J_E value of sedoheptulose 1,7-bisphosphatase (SBPase) was almost one under a wide range of conditions (Harrison et al., 1998). In contrast, although phosphoribulokinase catalyzes a virtually irreversible reaction in the PCR cycle, its C^J_E was near zero until the enzyme level in transgenic tobacco plants was reduced to 20% of wild type (Paul et al., 1995). Reduction in aldolase levels caused a severe decrease in photosynthesis, with the activities of FBPase and SBPase showing a proportional reduction in transgenic potato plants (Haake et al., 1998, 1999). The C^J_E value of transketolase was also near unity (Henkes et al., 2001). Aldolase and transketolase catalyze reversible reactions in the PCR cycle, but their activities in chloroplasts are no greater than the demand exerted by photosynthesis. Those enzymes functioning with rate-limiting activities in the PCR cycle could become targets for the genetic manipulation of crops with the aim of improving the photosynthetic performance of essential reactions in primary carbon fixation pathways.