Kinetic understanding of the T. brucei trypanothione synthesis pathway

SysMO is a European transnational funding and research initiative on "Systems Biology of Microorganisms".

The goal pursued by SysMO was to record and describe the dynamic molecular processes going on in unicellular microorganisms in a comprehensive way and to present these processes in the form of computerized mathematical models.

Systems biology will raise biomedical and biotechnological research to a new quality level and contribute markedly to progress in understanding. Pooling European research ...

The SilicoTryp project aims at the creation of a “Silicon Trypanosome”, a comprehensive, experiment-based, multi-scale mathematical model of trypanosome physiology. Trypanosomes are blood-stream parasites transmitted by tsetse flies; they cause African sleeping sickness in humans and livestock. Currently available drugs have severe side effects, and the parasites are rapidly developing resistance. In this project, we collect a wide range of new experimental data on the parasite in its various ...

The enzyme Trypanothione Synthetase (TryS) is a complex enzyme that catalyses the two step reaction that forms trypanothione from 2 molecules of GSH and 1 molecule of Spd and the use of ATP

Parasites will be harvested at different growth phases and the total amount of the proteins will be followed by western blot. The absolut concentration will be obtained by comparison with a know amount of the recombinant untagged protein. The thiol redox state of the proteins will be followed by modification of the free cys with methoxy-ethyl-maleinimide poly(ethylenglycol) (Meo-PEG-mal).

The enzymes involved in the trypanothione metabolism will be studied in a uniform assay medium that mimics the intracellular milieu of the parasite.

This assay is designed to obtain the in vitro kinetic data of T. brucei recombinant trypanothione synthetase. The enzyme catalyzes the ATP-dependent ligation of spermidine (Spd) and GSH to generate glutathionylspermidine (Gsp) and also of Gsp and GSH to finally produce trypanothione (T(SH)2). The data was obtained in an spectrophotometric assay that links ADP production with NADH consumption through the piruvte kinase and lactate dehydrogenase.

We here create a kinetic model for a single enzyme within the T. brucei trypanothione synthesis pathway, the enzyme trypanothione synthetase based on the insights from the laboratory experiments

TbTryS activity was measured at 37°C in the in vivo-like buffer. All substrate stock solutions were prepared in the in vivo-like buffer and the pH was adjusted to 7.0. The assays were performed in a final volume of 2 ml and contained 0.2 mM NADH, 1 mM phosphoenolpyruvate, 4 units pyruvate kinase, 4 units L-lactate dehydrogenase, 0.17 µM TbTryS, 2.1 mM ATP and varying amounts of glutathione, and spermidine.

All datapoints that were measured are displayed together with the accompanying simulations by the computational model

TbTryS activity was measured at 37°C in the in vivo-like buffer. All substrate stock solutions were prepared in the in vivo-like buffer and the pH was adjusted to 7.0. The assays were performed in a final volume of 2 ml and contained 0.2 mM NADH, 1 mM phosphoenolpyruvate, 4 units pyruvate kinase, 4 units L-lactate dehydrogenase, 0.17 µM TbTryS, 2.1 mM ATP and varying amounts of GSH, and Spd.

An extended model description of the TryS model

The file contains the initial rate measurements of TbTryS obtained under different substrate and product initial concentrations.

Mechanistical model of the catalytic cycle of Trypanothione Synthetase

This method describes how to derivatize the N-glutathionylspermidine and trypanothione produced by T. brucei trypanothione synthetase under in vivo-like conditions

This is a protocol for determining the activity of the T. brucei Trypanothione synthetase under in vivo-like conditions.

This method describes the preparation of the in vivo-like buffer for the measurement of bloodstream T. brucei recombinant enzymes under pseudo-physiological conditions.