A method to study complex enzyme kinetics involving numerical analysis of enzymatic schemes. The mannitol permease of Escherichia coli as an example.
An analysis of complex kinetic mechanisms is proposed that consists of two steps, (i) building of an kinetic scheme from experimental data other than steady-state kinetics and (ii) numerical simulation and analysis of the kinetics of the proposed scheme in relation to the experimental kinetics. Procedures are introduced to deal with large numbers of enzymatic states and rate constants, and numerical tools are defined to support the analysis of the scheme. The approach is explored by taking the mannitol permease of Escherichia coli as an example. This enzyme catalyzes both the transport of mannitol across the cytoplasmic membrane and the phosphorylation of mannitol. The challenge is to deduce the transport properties of this dimeric enzyme from the phosphorylation kinetics. It is concluded that (i) the steady-state kinetic behavior is largely consistent with the proposed catalytic cycle of the monomeric subunit, (ii) the kinetics provide no direct support but also do not disprove a coupled translocation of the binding sites on the two monomeric subunits. The approach reveals the need for further experimentation where the implementation of experimental results in the scheme conflict with the experimental kinetics and where specific experimental characteristics do not show up in the simulations of the proposed kinetic scheme.
Lolkema
J S
JS
Department of Microbiology, University of Groningen, Haren, The Netherlands.
D001665:Binding Sites; D004798:Enzymes / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008961:Models, Structural; D008962:Models, Theoretical; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010766:Phosphorylation
D004798:Enzymes; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli