Control of the Reactivation Kinetics of Homodimeric Triosephosphate Isomerase from Unfolded Monomers

Abstract

Homodimeric triosephosphate isomerases from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have markedly similar catalytic properties and 3-D structures; their overall amino acid sequence identity is 68% and 85% in their interface residues. Nonetheless, active dimer formation from guanidinium chloride unfolded monomers is faster and more efficient in TcTIM than in TbTIM. The enzymes thus provide a unique opportunity for exploring the factors that control the formation of active dimers. The kinetics of reactivation at different protein concentrations showed that the process involved three reactions: monomer folding, association of folded monomers, and a transition from inactive to active dimers. The rate constants of the reactions indicated that, at relatively low protein concentrations, the rate-limiting step of reactivation was the association reaction; at high protein concentrations the transition of inactive to active dimers was rate limiting. The rates of the latter two reactions were higher in TcTIM than in TbTIM. Studies with a mutant of TcTIM that had the interface residues of TbTIM showed that the association rate constant was similar to that of TbTIM. However, the rate of the transition from inactive to active dimers was close to that of TcTIM; thus, this transition depends on the noninterfacial portion of the enzymes. When unfolded monomers of TcTIM and TbTIM were allowed to reactivate together, TcTIM, the hybrid, and TbTIM were formed in a proportion of 1:0.9:0.2. This distribution suggests that, in the hybrid, the characteristics of the TcTIM monomers influence the properties of TbTIM monomers.

New chemotypes as Trypanosoma cruzi triosephosphate isomerase inhibitors: a deeper insight into the mechanism of inhibition

“…In a second step of the experiment, the denaturing mixture was diluted in buffer containing different concentrations of the studied compounds. As previously shown by Zomosa-Signoret et al 22 , the removal of the denaturant, GdnHCl, by dilution leads to the refolding and association of the monomers to form the catalytically active dimer. This reaction can be followed, by measuring the appearance of activity through time.…”

In Vitro Refolding of Triosephosphate Isomerase from L. donovani

“…3). In the absence of BSA, and in consonance with other works [17][18][19], we observed that the extent of reactivation increased with protein concentration indicating that the rate of reaction 2 is central in the reactivation of LdTIM.…”

1,2,4-thiadiazol-5(4H)-ones: a new class of selective inhibitors of Trypanosoma cruzi triosephosphate isomerase. Study of the mechanism of inhibition

“…In a second step of the experiment, the denaturing mixture was diluted in buffer containing different concentrations of the compound. As previously shown by 24 , the removal of the denaturant by dilution leads to the refolding and association of the monomers to form the catalytically active dimer. This reaction can be followed by measuring the appearance of activity through time.…”