Inhibition of Leishmania infantum trypanothione reductase by NADPH, its natural substrate

Trypanothione reductase is a flavoprotein that catalyzes the β-NADPH-linked reduction of trypanothione (N1,N8-bis (glutathionyl)spermidine), a glutathione analog unique to trypanosomatid parasites [1]. The atomic structure of this enzyme from Leishmania infantum has been solved [2]. The enzyme contains a FAD moiety, a pair of redox-active cysteine residues (52 & 57) involved in trypanothione reduction, and two distinct binding sites for NADPH and trypanothione. Single- or double-mixing stopped-flow experiments in which trypanothione reductase is pre-mixed with either trypanothione or NADPH, indicate that turnover efficiency is critically dependent on the order of substrate addition. When trypanothione is allowed to occupy its binding site first, the enzyme obeys standard Michaelis-Menten kinetics with steady-state parameters kCAT = 68.0  0.2 s1 and KM for NADPH = 1.5  0.01 M. On the contrary, if the enzyme is pre-mixed with NADPH, which determines the formation of a characteristic charge-transfer complex between the FAD moiety and the proximal active-site sulfhydryl of Cys57, the enzyme enters a catalytically incompetent state whose lifetime depends on several factors. Preliminary Maldi-Tof mass spectrometry experiments performed on this NADPH-inactivated enzyme indicate that the mass of trypanothione reductase is increased by an amount consistent with the weight of trypanothione, an indication that trypanothione may have crosslinked to the polypeptide chain, at a position which is currently under investigation. Alltogether these results suggest that trypanothione reductase may undergo an auto-modification reaction during turnover allowing for the allosteric regulation of its enzymatic activity.