Analysis of the Hierarchical and Metabolic Regulation of Flux Through Metabolic Pathways

Abstract

Bioremediation strategies are often based on empirical approaches. Enhanced understanding of the regulation of metabolic flux through the metabolic networks of oil-degrading microorganisms will benefit predicting and rationally directing microbial activities, such that one can better deal with oil pollution. Hierarchical regulation analysis (HRA) quantifies how perturbation changes in vivo flux through a metabolic network, at the level of the individual enzymes participating in these fluxes. It reveals for each investigated enzyme the degree to which the change in flux through the enzyme is due to hierarchical regulation (change in gene expression, i.e., maximum enzyme activity, expressed as the hierarchical regulation coefficient) and metabolic regulation (changes in the interaction of enzymes with substrates, products, or allosteric effectors through changes in concentrations of the latter, expressed as the metabolic regulation coefficient). Measuring enzyme levels and in vivo fluxes through those enzymes, under two or more different conditions, is sufficient to determine the coefficients. The slope in a double logarithmic plot of the concentrations of an enzyme against the corresponding flux through this enzyme provides the hierarchical regulation coefficient. On the basis of a simple mathematical concept derived from enzyme kinetics, one can subsequently calculate the metabolic regulation coefficient. A detailed general protocol on how to design and execute HRA experiments is provided. The protocol considers all steps between culturing the microorganisms and interpreting the determined regulation coefficients: the growth conditions, protein extraction, determination of specific enzyme activities, determining flux, calculation of the regulation coefficients, and interpretation of these coefficients.