Metabolic control analysis provides a robust mathematical and theoretical framework for describing metabolic and signaling pathways and networks, and for quantifying the controls over these processes. It can deal with systems of any complexity or architecture and does not require all system components to be known a priori, making it a valuable post-genomic tool. It was developed in the 1970s by Kacser and Burns [18] and Heinrich and Rapoport [19]. Since then, dedicated researchers have expanded and advanced metabolic control analysis theory and applications, carefully defined the associated terms, and developed analytical and educational tools [20,21,22,23,24,25].

Metabolic control analysis uses equations based on the kinetics of enzymes (known as elasticity coefficients) to parameterize control coefficients (which describe the degree of control exerted by any given component on a particular output) resulting in response coefficients. The response to a perturbation is quantified by the summation of the response coefficients affecting the output of interest, which depends on both the control coefficients (systems properties) and elasticity coefficients (local properties). The fundamental equations are presented in Box 1 (also see Figure 1). It should be noted, explicitly, that the use of metabolic control analysis is not limited to linear pathways, but is also applicable to branching and cyclic pathways, and enzyme cascades.

Metabolic control analysis can be used to quantitate control exerted by different components of a specific system and pinpoint areas requiring further experimentation. For example, Krauss and Brand [17] recently used metabolic control analysis to quantitate the contributions of known and unknown signal transduction pathways in the early response of thymocytes to mitogen (concanavalin A, ConA) stimulation. To apply metabolic control analysis to a complex system containing both metabolic and signaling components, they gathered large parts of metabolism or signal transduction into 'black-box' groups of reactions, coupled signal transduction events to cellular variables, and limited the time frame of observation. In particular, thymocyte response to Con A stimulation was quantified by measuring steady-state respiration rates, and signaling routes (such as protein kinase C (PKC)) were grouped based on their sensitivity to specific inhibitors. The effects of these known and unknown signal transduction pathways on the mitochondrial membrane potential, a key intermediate in respiration, were also quantified. The analysis of the model system, presented in Figure 2, resulted in a quantitative topology of signaling routes involved in the early phase of mitogen stimulation of thymocytes. Novel findings, such as the significant role played by calcineurin signal transduction pathways (30% of total), highlight areas for future experimental work.

The application of metabolic control analysis has altered our basic understanding of metabolic control. In particular, the belief that control over a pathway is dictated by a 'rate-limiting step' is now obsolete and is being removed from the biochemistry textbooks. Instead, it is replaced by the concept of shared control, where many - or theoretically all - enzymes in a pathway have a role in controlling the flux through the pathway. Implicit in this is the idea that the regulation of cell metabolism requires coordinate change in the activities of many enzymes ('multisite modulation' [26]). The validity of this notion has been supported by bioengineers' lack of success in increasing a particular flux (product yield) by overexpressing the 'rate-limiting' enzyme and success by overexpressing a group of enzymes in a pathway. For example, Niederberger et al. [27] found that overexpression of four of the five enzymes in the yeast biosynthetic pathway leading from chorismate to tryptophan was required to significantly increase (more than eightfold) the production of tryptophan.

If the coordinated expression of enzymes in a pathway is required to significantly increase its flux, this should be an underlying design principle of organisms. In fact, the coordinated induction of enzymes to increase metabolic flux through a pathway has long been observed in vivo. One of many examples provided by Fell [21] is the urea synthetic pathway. The rate of urea synthesis in rats responds proportionately to the amount of protein in the diet. When rats are fed on diets that increase urea output fourfold, eight of the enzymes measured increased significantly, including all four of the urea-cycle enzymes [28]. With DNA microarrays and complete genome information, global expression data detailing the coordinated induction of pathway enzymes may be coupled with structural information on the organization of genes for pathway enzymes in operons or in clusters with common cis-acting elements (for example, see [29]). By performing these types of analyses on organisms responding to a variety of external effectors (such as nutrient conditions, pathogens, and so on), and on diverse organisms, this underlying design principle may be further explored.

Metabolic control analysis can also be used to explain why most mutations in diploid organisms are 'fully' recessive. Most enzymes have low-flux control coefficients; thus, a 50% reduction in enzyme concentration resulting from a null mutation in one allele of a diploid pair has little effect on the pathway flux. In addition, because pathway flux is a systems property, the influence of an alteration at one locus is measured in the whole system, minimizing the impact from any one reduction. Kacser and Burns [30] therefore posited the phenomenon of genetic dominance as the "inevitable consequence of the kinetic structure of enzyme networks" and not a result of natural selection. This conclusion was supported by Orr [31], who found the same extent of recessive mutations in artificial diploids created from the haploid organism Chlamydomonas reinhardtii (where the possibility of selection in the diploid was eliminated). The existence of a limited number of 'partially' recessive mutants, in which the heterozygote has an intermediate phenotype, is also consistent with metabolic control analysis. Theoretically, these enzymes (with high control coefficients) would be more likely to be a part of a very small pathway or the first enzyme of a branching pathway. Despite these studies, the 'inevitability' of dominance is still debated [32]. With the availability of complete genome information for a number of diploid organisms, genomic information on natural variants (for example, the Arabidopsis ecotypes Columbia and Landsberg [33]), and numerous collections of mutants, this type of question can now be addressed on a global scale.