USING EVOLUTIONARY CHANGES TO ACHIEVE SPECIES-SPECIFIC INHIBITION OF ENZYME ACTION - STUDIES WITH TRIOSEPHOSPHATE ISOMERASE

Background: Many studies that attempt to design species-specific drugs focus on differences in the three-dimensional structures of homologou s enzymes. The structures of homologous enzymes are generally well con served especially at the active site, but the amino-acid sequences are often very different. We reasoned that ii a non-conserved amino acid is fundamental to the function or stability of an enzyme from one part icular species,one should be able to inhibit only the enzyme from that species by using an inhibitor targeted to that residue. We set out to test this hypothesis in a model system. Results: We first identified a non-conserved amino acid (Cys14) whose integrity is important for ca talysis in triosephosphate isomerase (TIM) from Trypanosoma brucei. Th e equivalent residues in rabbit and yeast TIM are Met and Leu, respect ively A Cys14Leu mutant of trypanosomal TIM had a tendency to aggregat e, reduced stability and altered kinetics. To model the effects of a m olecule targeted to Cys14, we used methyl methanethiosulfonate (MMTS) to derivatize Cys14 to a methyl sulfide. This treatment dramatically i nhibited TIMs with a Cys residue at a position equivalent to Cys14, bu t not rabbit TIM (20% inhibition) or yeast TIM (negligible inhibition) , which lack this residue. Conclusions: Cys14 of trypanosomal TIM is a non-conserved amino acid whose alteration leads to loss of enzyme str ucture and function. TIMs that have a cysteine residue at position 14 could be selectively inhibited by MMTS. This approach may offer an alt ernative route to species-specific enzyme inhibition.