An integrated study of threonine-pathway enzyme kinetics in Escherichia coli

Abstract

We have determined the kinetic parameters of the individual steps of the threonine pathway from aspartate in Escherichia coli under a single set of experimental conditions chosen to be physiologically relevant. Our aim was to summarize the kinetic behaviour of each enzyme in a single tractable equation that takes into account the effect of the products as competitive inhibitors of the substrates in the forward reaction and also, when appropriate (e.g. near-equilibrium reactions), as substrates of the reverse reactions. Co-operative feedback inhibition by threonine and lysine was also included as necessary. We derived the simplest rate equations that describe the salient features of the enzymes in the physiological range of metabolite concentrations in order to incorporate them ultimately into a complete model of the threonine pathway, able to predict quantitatively the behaviour of the pathway under natural or engineered conditions.

Control of the threonine-synthesis pathway in Escherichia coli: a theoretical and experimental approach

“…This was because the enzyme was approaching equilibrium near the end of the time course on account of the accumulation of its product phosphate formed by the hydrolysis of ATP. By trial and error, a value of 1.78i10' was found to be suitable, which was higher than our experimentally determined value (Table 2 and [20]), but still lower than the previously published value [34]. The second change related to the endogenous consumption of ATP by the extract, where it seemed that the experimental measurement in the absence of the other metabolites and net threonine synthesis appeared to be an over-estimate in their presence.…”

“…All chemicals, the preparation of crude extract and the enzyme assays are described in the accompanying papers [20,21].…”

“…The development of the enzyme rate expressions and the measurement of their parameters has been described in the first paper of this series [20]. Except where otherwise stated, the simulations reported here use those values (see Table 2).…”

“…It is for these reasons that in the previous two papers [20,21] we have described the experimental measurements that have formed the basis for the development of the model of E. coli threonine synthesis described here. The model functions in two ways : it can predict the time course of threonine synthesis for particular levels of enzymes and intermediates of central metabolism, and it can predict the flux control coefficients of the enzymes under a range of intracellular conditions different from those of the original measurements.…”

Mathematical modelling of aliphatic glucosinolate chain length distribution in Arabidopsis thaliana leaves

“…Heinrich et al 1977;Galazzo and Bailey 1990;Heinrich and Schuster 1996;Chassagnole et al 2001). Accurate in vivo kinetic data for glucosinolate biosynthetic enzymes are virtually impossible to obtain in our present state of knowledge because measurement would need to be restricted to only those cells involved in glucosinolate production.…”