Factors That Control the Reactivity of the Interface Cysteine of Triosephosphate Isomerase from Trypanosoma brucei and Trypanosoma cruzi

Abstract

The amino acid sequences and X-ray structures of homodimeric triosephosphate isomerase from the pathogenic parasites Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM) are markedly similar. In the two TIMs, the side chain of the only interface cysteine (Cys14) of one subunit docks into loop 3 of the other subunit. This portion of the interface is also markedly similar in the two enzymes. Nonetheless, Cys14 of TcTIM is nearly 2 orders of magnitude more susceptible to the thiol reagent methylmethane thiosulfonate (MMTS) than Cys14 of TbTIM. The causes of this difference were explored by measuring the second-order rate constant of inactivation by MMTS (k(2)) under various conditions. At pH 7.4, k(2) in TcTIM is 70 times higher than in TbTIM. The difference decreases to 30 when the amino acid sequence of loop 3 and adjoining residues of TbTIM are conferred to TcTIM (triple mutant). The pK(a) values of the thiol group of the interface cysteine of TcTIM and the triple mutant were 0.7 pH unit lower than in TbTIM. Because this difference could account for the different sensitivity of the enzymes to thiol reagents, we determined the k(2) of inactivation at equal levels of ionization of their interface cysteines. Under these conditions, the difference in k(2) between TcTIM and TbTIM became 8-fold, whereas that of the triple mutant to TbTIM was 1.5 times. The substrate analogue phosphoglycolate did not modify the pK(a) of the thiol group of the interface, albeit it diminished the rate of its derivatization by MMTS. In the presence of phosphoglycolate, under conditions in which the interface cysteines of the enzymes had equal levels of protonation, the difference in k(2) of TcTIM and TbTIM became smaller, whereas k(2) of the triple mutant was almost equal to that of TbTIM. Thus, from measurements of the reactivity of the interface cysteine in various conditions, it was possible to obtain information on the factors that control the dynamics of a portion of the dimer interface.

Identification of the critical residues responsible for differential reactivation of the triosephosphate isomerases of two trypanosomes

“…In this work, we investigated which regions and which amino acids are responsible for these differences. This investigation was based on previous observations that have shown that in TIM only native dimers have catalytic activity and this is considered to be evidence that the quaternary structure of the enzyme is correct [8,9,23,24]. Even though there are also reports of activities in monomeric TIMs, these activities are very much lower than those reported for TbTIM and TcTIM [25,26].…”

Structural Basis for Redox Regulation of Cytoplasmic and Chloroplastic Triosephosphate Isomerases from Arabidopsis thaliana

“…Our steady-state kinetic characterization indicates that cTPI catalyzes the G3P to DHAP isomerization with a K m of 0.48 mM and a turnover number (k cat ) of 2565 s −1 , corresponding to a catalytic efficiency of 5.34 × 10 6 M −1 s −1 ; whereas pdTPI displayed a K m of 0.40 mM and a k cat of 2337 s −1 , corresponding to a catalytic efficiency of 5.48 × 10 6 M −1 s −1 . The observed K m values are comparable to constants from other plant TPIs, such as pea seed TPI, with a value of 0.44 mM (Turner et al, 1965), rye cTPI with a value of 0.6 mM, and its chloroplast counterpart with a value of 0.68 mM (Kurzok and Feierabend, 1984), spinach pdTPI (Tang et al, 1999) and TPIs from other organisms such as P. falciparum (Samanta et al, 2011) S. cerevisiae (Gonzalez-Mondragon et al, 2004), and T. cruzi (Reyes-Vivas et al, 2001; Table 1). In our hands pdTPI presents a k cat of 2337 s −1 that contrasts with the value of 424 s −1 previously reported (Chen and Thelen, 2010).…”

A new approach for potential drug target discovery through in silico metabolic pathway analysis using Trypanosoma cruzi genome information

“…The triose phosphate isomerase (TIM) is also a central enzyme of the glycolytic pathway that has been studied in T. cruzi and in a number of pathogenic protozoa (Pérez-Montfort et al 1999, Reyes-Vivas et al 2001, Rodríguez-Romero et al 2002, Olivares-Illana et al 2006. Recently, two studies showed the possibility of developing new therapeutic agents against trypanosomes using TIM as a molecular target.…”