title: Species-specific inhibition of Giardia lamblia triosephosphate isomerase by localized perturbation of the homodimer.
abstract: Giardia lamblia depends on glycolysis to obtain ATP, highlighting the suitability of glycolytic enzymes as targets for drug design. We studied triosephosphate isomerase from G. lamblia (GlTIM) as a potential species-specific drug target. Cysteine-reactive agents were used as probes, in order to test those regions near to cysteine residues as targets to perturb enzyme structure and activity. Methyl methanethiosulfonate (MMTS) derivatized three of the five Cys per subunit of dimeric GlTIM and induced 50% of inactivation. The 2-carboxyethyl methanethiosulfonate (MTSCE) modified four Cys and induced 97% of inactivation. Inactivation by MMTS or MTSCE did not affect secondary structure, nor induce dimer dissociation; however, Cys modification decreased thermal stability of enzyme. Inactivation and dissociation of the dimer to stable monomers were reached when four Cys were derivatized by 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). The effects of DTNB were completely abolished when GlTIM was first treated with MMTS. The effect of thiol reagents on human TIM was also assayed; it is 180-fold less sensitive than GlTIM. Collectively, the data illustrate GlTIM as a good target for drug design.
authors: Laboratorio de Bioquímica-Genética y Dirección de Investigación, Instituto Nacional de Pediatría, 04530 México, DF, Mexico.
authors: Sergio
authors: S
authors: Enriquez-Flores
authors: Adela
authors: A
authors: Rodriguez-Romero
authors: Gloria
authors: G
authors: Hernandez-Alcantara
authors: Ignacio
authors: I
authors: De la Mora-De la Mora
authors: Pedro
authors: P
authors: Gutierrez-Castrellon
authors: Karla
authors: K
authors: Carvajal
authors: Gabriel
authors: G
authors: Lopez-Velazquez
authors: Horacio
authors: H
authors: Reyes-Vivas
mesh_terms: D000818:Animals; D003545:Cysteine / Q000378:metabolism; D019281:Dimerization; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004795:Enzyme Stability; D016829:Giardia lamblia / Q000201:enzymology*; D006358:Hot Temperature; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors*; D014305:Triose-Phosphate Isomerase / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: D015800:Protozoan Proteins; C014674:methyl methanethiosulfonate; D004228:Dithionitrobenzoic Acid; D008741:Methyl Methanesulfonate; D014305:Triose-Phosphate Isomerase; D003545:Cysteine