Sulfhydryl reagent susceptibility in proteins with high sequence similarity

Abstract

The amino acid sequence of triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana have an identity of 68%. Using the numbering system for the T. brucei enzyme, in their aligned sequences, the T. cruzi and leishmanial enzymes have cysteine residues at positions 14, 40, 117 and 126. T. brucei triosephosphate isomerase has cysteine residues at positions 14, 40 and 126, and a valine residue at position 117. Dithionitrobenzoic acid and methylmethane thiosulfonate inhibited the three enzymes, but T. cruzi triosephosphate isomerase was more than 100-fold more sensitive. The sensitivity of wild type triosephosphate isomerase from T. cruzi and T. brucei to the reagents was equal to that of the Cys117Val and Val117Cys mutant enzymes, respectively. Triosephosphate isomerases that have cysteine residues at positions 40 and 126, but lack a cysteine residue at position 14 are insensitive to methylmethane thiosulfonate. Thus, sulfhydryl reagents act on Cys14. At stoichiometric concentrations, the reagents inhibited the three enzymes as a consequence of structural alterations as measured by binding of 8-anilino-1-napthalenesulfonic acid to previously buried hydrophobic regions. However, the times for half-maximal alterations were 10 min, 15 hours and over 30 hours for T. cruzi, T. brucei and L. mexicana triosephosphate isomerase, respectively. The effect of pH on the action of the sulfhydryl reagents and molecular modeling showed no differences in the solvent accesibility of Cys14. As Cys14 forms part of the dimer interface, the data indicate that, in the three enzymes, barriers of different magnitude hinder the interaction between the sulfhydryl reagents and Cys14. The barrier is lower in T. cruzi triosephosphate isomerase which makes its dimer interface more susceptible for perturbation.Keywords : triosephosphate isomerase; trypanosome ; cysteine residue; protein conformations; protein changes.Triosephosphate isomerase is a homodimeric enzyme whose activity. Some attempts have been made with this target in mind kinetics [1] and structure [2Ϫ8] are well established. It is the [20]. prototype of an A-β barrel and thus, this enzyme has been extenOur group has worked on the hypothesis that nonconserved sively studied with respect to the relation that exists between its amino acids that are central for stability or catalysis can be structure and function [9Ϫ11]. It is also noteworthy that excellent targets for achieving species-specific inhibition of triosephosphate isomerase has been considered a potential target homologous enzymes [21]. Through site-directed mutagenesis for drug design against pathogenic parasites [12Ϫ14] ; the latter and chemical-modification studies, we found that the integrity studies have focused on the active center of the enzyme. Studies of the nonconserved Cys14 of triosephosphate isomerase from on the reactivation of triosephosphate isomerase from denatured Trypanosoma brucei is fundamental for maintaining its structure triosephosphate isomerase monomers [15Ϫ17] ha...

Metabolic reconfiguration of the central glucose metabolism: a crucial strategy ofLeishmania donovanifor its survival during oxidative stress

“…4 and 5), but specific activity determination of TPI showed a downregulation following treatment with menadione and diamide (Table 1). This observation correlated with the study that activity of TPI is sensitive toward sulfhydryl reagents due to presence of cysteine residues at 14, 40, 117, and 146 (51). Interestingly PGI, the cytosolic counterpart of glycolytic pathway specific to Leishmania showed a timedependent up-regulation (Fig.…”

Disulfide Bridges in the Mesophilic Triosephosphate Isomerase from Giardia lamblia Are Related to Oligomerization and Activity

“…7 The enzymes of this metabolic pathway have been proposed as potential targets for drug design in other parasites. [9][10][11][12][13] Recently, we characterized a recombinant triosephosphate isomerase (EC 5.3.1.1) from G. lamblia (GlTIM). 13 This enzyme catalyzes the interconversion between D-glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP).…”

Crystal structure of triosephosphate isomerase from Trypanosoma cruzi in hexane

“…Several lines of evidence indicate that the interactions of the side chain of residue 15 with loop 3 are central in dimer stability and enzyme catalysis (39)(40)(41); along this line, it is noted that the derivatization of Cys-15 by thiol reagents produces drastic irreversible structural alterations and abolition catalysis (39,40). Because human TIM has a methionine in position 15, it has been suggested that this region of the enzyme can be targeted for species specific inhibition of TcTIM (23,39,40). In fact, TcTIM is completely inhibited by sulfhydryl reagents at concentrations that hardly affect human TIM or TIMs that lack a cysteine in position 15 (42).…”