CLONING, EXPRESSION, PURIFICATION AND CHARACTERIZATION OF TRIOSEPHOSPHATE ISOMERASE FROM TRYPANOSOMA-CRUZI

The gene that encodes for triosephosphate isomerase from Trypanosoma c ruzi was cloned and sequenced. In T. cruzi, there is only one gene for triosephosphate isomerase. The enzyme has an identity of 72% and 68% with triosephosphate isomerase from Trypanosoma brucei and Leishmania mexicana, respectively. The active site residues are conserved: out of the 32 residues that conform the interface of dimeric triosephosphate isomerase from T. brucei, 29 an conserved in the T. cruzi enzyme. The enzyme was expressed in Escherichia coli and purified to homogeneity. Data from electrophoretic analysis under denaturing techniques and fi ltration techniques showed that triosephosphate isomerase from T. cruz i is a homodimer. Some of its structural and kinetic features were det ermined and compared to those of the purified enzymes from T. brucei a nd L. mexicana. Its circular dichroism spectrum was almost identical t o that of triosephosphate isomerase from T. brucei. Its kinetic proper ties and pH optima were similar to those of T. brucei and L. mexicana, although the latter exhibited a higher V-max with glyceraldehyde 3-ph osphate as substrate. The sensitivity of the three enzymes to the sulf hydryl reagent methylmethane thiosulfonate (MeSO(2)-SMe) was determine d; the sensitivity of the T. cruzi enzyme was about 40 times and 200 t imes higher than that of the enzymes from T. brucei and L. mexicana, r espectively. Triosephosphate isomerase from T. cruzi and L. mexicana h ave the three cysteine residues that exist in the T. brucei enzyme (po sitions 14, 39, 126, using the numbering of the T. brucei enzyme); how ever, they also have an additional residue (position 117). These data suggest that regardless of the high identity of the three trypanosomat id enzymes, there are structural differences in the disposition of the ir cysteine residues that account for their different sensitivity to t he sulfhydryl reagent. The disposition of the cysteine in triosephosph ate isomerase from T. cruzi appears to make it unique for inhibition b y modification of its cysteine.