SULFHYDRYL REAGENT SUSCEPTIBILITY IN PROTEINS WITH HIGH SEQUENCE SIMILARITY TRIOSEPHOSPHATE ISOMERASE FROM TRYPANOSOMA-BRUCEI, TRYPANOSOMA-CRUZI AND LEISHMANIA-MEXICANA

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 al igned sequences, the T. cruzi and leishmanial enzymes have cysteine re sidues at positions 14, 40, 117 and 126, T. brucei triosephosphate iso merase has cysteine residues at positions 14, 40 and 126, and a valine residue at position 117. Dithionitrobenzoic acid and methylmethane th iosulfonate inhibited the three enzymes, but T. cruzi triosephosphate isomerase was more than 100-fold more sensitive. The sensitivity of wi ld type triosephosphate isomerase from T. cruzi and T. brucei to the r eagents was equal to that of the Cys117Val and Val117Cys mutant enzyme s, respectively. Triosephosphate isomerases that have cysteine residue s at positions 40 and 126, but lack a cysteine residue at position 14 are insensitive to methylmethane thiosulfonate. Thus, sulfhydryl reage nts act on Cys14. At stoichiometric concentrations, the reagents inhib ited the three enzymes as a consequence of structural alterations as m easured by binding of 8-anilino-1-napthalenesulfonic acid to previousl y buried hydrophobic regions. However, the times for half-maximal alte rations were 10 min, 15 hours and over 30 hours for T. cruzi, T. bruce i 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 f orms part of the dimer interface, the data indicate that, in the three enzymes, barriers of different magnitude hinder the interaction betwe en the sulfhydryl reagents and Cys14. The barrier is lower in T. cruzi triosephosphate isomerase which makes its dimer interface more suscep tible for perturbation.