Kinetic analysis and computational modelling of Trypanosoma brucei trypanothione synthetase

LEROUX, ALEJANDRO E.; HAANSTRA, J. R.; BAKKER, B. M.; KRAUTH-SIEGEL, R. LUISE

Ruedesheim/Rhein

Simposio; 6th International Beilstein Symposium; 2013

Beilstein Institute

Trypanosoma brucei is the causative agent of human African trypanosomiasis, one of the most important neglected tropical diseases. The parasite possesses a unique redox metabolism which is based on the dithiol trypanothione [T(SH)2]. This bis(glutathionyl)spermidine conjugate delivers the electrons for the synthesis of DNA precursors, the detoxification of hydroperoxides, and other thiol-dependent pathways. African trypanosomes employ trypanothione synthetase (TryS) as a single enzyme to generate both glutathionylspermidine (Gsp) and T(SH)2. In vitro, the enzyme also displays an amidase activity and in total can catalyse five different reactions: ATP hydrolysis, formation of Gsp and T(SH)2 as well as hydrolysis of the conjugates to regenerate GSH and spermidine. We have done a thorough kinetic analysis of T. brucei TryS in a newly developed buffer system at pH 7.0 and 37 °C, mimicking the physiological environment of the enzyme in the cytosol of the parasites. TryS shows apparent Km-values for GSH, ATP, spermidine and Gsp of 34, 18, 687, and 32 µM, respectively, as well as Ki-values for GSH and T(SH)2 of 1 mM and 360 µM, respectively. To obtain a deeper insight in the molecular mechanism of TryS, we have formulated alternative kinetic models, with elementary reaction steps represented by linear kinetic equations. The model parameters were fitted to the extensive matrix of steady-state data obtained under the in vivo-like conditions. The best model describes the full kinetic profile and is able to predict time course data that were not used for fitting. This systems biology approach to enzyme kinetics led us to conclude that (i) TryS follows a ter-reactant mechanism, (ii) the intermediate Gsp dissociates from the enzyme between the two catalytic steps and (iii) T(SH)2 inhibits the enzyme by remaining bound at its product site and, as does the inhibitory GSH, by binding to the activated enzyme complex.