Dissecting the catalytic mechanism of Trypanosoma brucei trypanothione synthetase by kinetic analysis and computational modellin

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

Frankfurt am Main

Congreso; International Symposium of the German Society for Biochemistry and Molecular Biology (GBM); 2013

German Society for Biochemistry and Molecular Biology (GBM)

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, methionine sulfoxide reduction and other thiol-dependent pathways. Trypanothione synthetase (TryS) catalyzes the synthesis of both glutathionylspermidine (Gsp) and T(SH)2. 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 which multiply in the blood of the mammalian host. TryS displays 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.