An unusual triosephosphate isomerase from the early divergent eukaryote Giardia lamblia

Abstract

Recombinant triosephosphate isomerase from the parasite Giardia lamblia (GlTIM) was characterized and immunolocalized. The enzyme is distributed uniformly throughout the cytoplasm. Size exclusion chromatography of the purified enzyme showed two peaks with molecular weights of 108 and 55 kDa. Under reducing conditions, only the 55-kDa protein was detected. In denaturing gel electrophoresis without dithiothreitol, the enzyme showed two bands with molecular weights of 28 and 50 kDa; with dithiotretitol, only the 28-kDa protein was observed. These data indicate that GlTIM may exist as a tetramer or a dimer and that, in the former, the two dimers are covalently linked by disulfide bonds. The kinetics of the dimer were similar to those of other TIMs. The tetramer exhibited half of the kcat of the dimer without changes in the Km. Studies on the thermal stability and the apparent association constants between monomers showed that the tetramer was slightly more stable than the dimer. This finding suggests the oligomerization is not related to enzyme thermostability as in Thermotoga maritima. Instead, it could be that oligomerization is related to the regulation of catalytic activity in different states of the life cycle of this mesophilic parasite.

Giardial Triosephosphate Isomerase as Possible Target of the Cytotoxic Effect of Omeprazole in Giardia lamblia

“…Although omeprazole modifies 1 of its 5 Cys in the same way as when we used DTNB (22), it does not inactivate HsTIM; indeed, it is almost 3,000 times more resistant than GlTIM. This is due to the properties of the region surrounding Cys 222 in GlTIM and its influence on the affinity for the substrate by the enzyme when such a residue is modified (24,27,31). The fluorescence of GlTIM-omeprazole adducts observed in recombinant enzymes (see Fig.…”

“…We used transformed Escherichia coli strain BL21(DE3)(pLysS) cells containing the wild-type hstim, wild-type gltim, and mutant gltim genes to produce the recombinant enzymes. Purification was performed as described previously (27), with the additional use of anionexchange (Q-Sepharose fast flow [FF]; 1.5 cm by 12 cm) and gel filtration (Superdex 75 prep grade; 1.6 cm by 60 cm) chromatography to obtain an enzyme purity of Ͼ95%. We calculated the protein concentrations of the mutants and wt-GlTIM by using the ε 280 of 26,600 M Ϫ1 cm Ϫ1 and ε 280 of 32,595 M Ϫ1 cm Ϫ1 for human triosephosphate isomerase (HsTIM).…”

“…Inactivation of recombinant enzymes. The activities of recombinant HsTIM and GlTIMs were determined in the direction of glyceraldehyde 3-phosphate (GAP) to dihydroxyacetone phosphate (DHAP) using a coupled system by following the decay of NADH at 340 nm and 25°C, as described elsewhere (27). The inactivation assays were performed at a protein concentration of 0.2 mg/ml (7.2 nM) in Tris-EDTA (TE) buffer (100 mM triethanolamine, 10 mM EDTA [pH 7.4]) at 37°C.…”

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).…”

Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus

“…Triosephosphate isomerase (TIM) is an important target for drug development Reyes-Vivas et al, 2001Zomosa-Signoret et al, 2003;Lopez-Velazquez et al, 2004) and vaccines (Zhu et al, 2006) directed against endoparasites. This enzyme participates in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate.…”