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 (kcat
) and/or expression or steady-state amount (km
) 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
= 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
closer to zero contributes little to the flux and an enzyme with CJE
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 CJE
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 CJE
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 CJE
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 CJE
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.