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 homologous enzymes. The structures of homologous enzymes are generally well conserved especially at the active site, but the amino-acid sequences are often very different. We reasoned that if a non-conserved amino acid is fundamental to the function or stability of an enzyme from one particular 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 catalysis in triosephosphate isomerase (TIM) from Trypanosoma brucei. The equivalent residues in rabbit and yeast TIM are Met and Leu, respectively. A Cys14Leu mutant of trypanosomal TIM had a tendency to aggregate, reduced stability and altered kinetics. To model the effects of a molecule targeted to Cys14, we used methyl methanethiosulfonate (MMTS) to derivatize Cys14 to a methyl sulfide. This treatment dramatically inhibited TIMs with a Cys residue at a position equivalent to Cys14, but 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 structure and function. TIMs that have a cysteine residue at position 14 could be selectively inhibited by MMTS. This approach may offer an alternative route to species-specific enzyme inhibition.
Gómez-Puyou
A
A
Departamento de Bioenergética, Universidad Nacional Autónoma de México, México DF.
Saavedra-Lira
E
E
Becker
I
I
Zubillaga
R A
RA
Rojo-Domínguez
A
A
Pérez-Montfort
R
R
D000818:Animals; D005075:Biological Evolution; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D007894:Leishmania mexicana / Q000201:enzymology; D008954:Models, Biological; D009154:Mutation; D011817:Rabbits; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D014305:Triose-Phosphate Isomerase / Q000037:antagonists & inhibitors*; D014346:Trypanosoma brucei brucei / Q000201:enzymology
D004791:Enzyme Inhibitors; D014305:Triose-Phosphate Isomerase; D003545:Cysteine