Computational protocol: Dissecting the Catalytic Mechanism of Trypanosoma brucei Trypanothione Synthetase by Kinetic Analysis and Computational Modeling*

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Protocol publication

[…] PySCeS (Python Simulator for Cellular Systems) open source modeling software () was used for initial model construction. Each elementary reaction step was described by reversible mass action kinetics, except the phosphorylation steps of GSH, which were modeled as irreversible. Alternative model versions were exported from PySCeS in sbml format to allow import into COPASI version 4.49.45 () for parameter estimation as well as steady-state and time course simulations. Constraints on the parameters were added to enforce microscopic reversibility in the central part of the model. For each face of the cubes (i.e. E-EA-EAB-EB-E) () the product of the rate constants going clockwise should equal the product of the rate constants going counterclockwise (). The concentrations in the model are in μm, and the time unit is in seconds. Fluxes of individual reactions within the model are in μm/s. For Spd as substrate, steady-state calculations were done at fixed concentrations of ATP, GSH, Spd, and T(SH)2. For Gsp, the procedure was the same except that the Gsp concentration was also fixed and that of Spd was zero. The model contains an irreversible reaction that consumes ADP, which mimics the coupling reaction in the enzymatic assay. In steady state, the rate of this reaction is equivalent to the overall ADP formation flux. For the steady-state simulations the total enzyme concentration, which is a conserved moiety, was set to 1 μm, and the calculated rates were normalized to enzyme concentration. Therefore, the overall ADP formation flux by the model is expressed in μm/s per 1 μm enzyme, leading to a unit of s−1. The time courses were simulated at 0.17 μm TryS, which was the enzyme concentration used in the corresponding experiment, with Gsp and T(SH)2 as variables. […]

Pipeline specifications

Software tools PySCeS, COPASI
Application Mathematical modeling
Organisms Trypanosoma brucei
Diseases Citrullinemia
Chemicals Adenosine Triphosphate, Spermidine