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Giardiasis, the infestation of the intestinal tract by Giardia lamblia, is one of the most prevalent parasitosis worldwide. Even though effective therapies exist for it, the problems associated with its use indicate that new therapeutic options are needed. It has been shown that disulfiram eradicates trophozoites in vitro and is effective in vivo in a murine model of giardiasis; disulfiram inactivation of carbamate kinase by chemical modification of an active site cysteine has been proposed as the drug mechanism of action. The triosephosphate isomerase from G. lamblia (GlTIM) has been proposed as a plausible target for the development of novel antigiardial pharmacotherapies, and chemical modification of its cysteine 222 (C222) by thiol-reactive compounds is evidenced to inactivate the enzyme. Since disulfiram is a cysteine modifying agent and GlTIM can be inactivated by modification of C222, in this work we tested the effect of disulfiram over the recombinant and trophozoite-endogenous GlTIM. The results show that disulfiram inactivates GlTIM by modification of its C222. The inactivation is species-specific since disulfiram does not affect the human homologue enzyme. Disulfiram inactivation induces only minor conformational changes in the enzyme, but substantially decreases its stability. Recombinant and endogenous GlTIM inactivates similarly, indicating that the recombinant protein resembles the natural enzyme. Disulfiram induces loss of trophozoites viability and inactivation of intracellular GlTIM at similar rates, suggesting that both processes may be related. It is plausible that the giardicidal effect of disulfiram involves the inactivation of more than a single enzyme, thus increasing its potential for repurposing it as an antigiardial drug., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mammalian triosephosphate isomerase spontaneously deamidates at Asn71 and Asn15 located at the subunit interface of the isologous dimer. These deamidations have been proposed to constitute the terminal marking event in the degradation of the enzyme. A series of physical and chemical studies detailed here reveals that the overall structure of the enzyme is substantially altered by these deamidations. The far-uv CD spectra show a 30% lower secondary structure with a blue shifted ellipticity minimum and increased fluorescence (10-22%) with a red-shifted emission maximum (8.7-15.6 nm) indicates exposure of tryptophans to a more polar environment. Increased binding of the fluorescent hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-disulfonic acid to the deamidated enzyme corroborates these spectral observations and also suggests that the hydrophobic residues at the subunit interface are exposed as a result of the deamidation. Decreased subunit cross-linking (80 vs 20%) of the deamidated enzyme by the bifunctional reagent ethylene glycolbis (succinimidylsuccinate) also indicates a loosening of the two subunits at the interface. These structural changes are accompanied by a decreased thermal stability (3.1 degrees C lower Tm) and an increased susceptibility to dissociation in urea. The terminal marking also results in the generation of new proteolytic sites and increases the susceptibility to proteolysis. Hybrid dimers from rabbit and yeast (lacking Asn71) showed that deamidation of the rabbit Asn71-yeast Asn15 pair does not accelerate deamidation of the remaining rabbit Asn15 site, indicating that deamidation of Asn71 is a prerequisite for deamidation of Asn15. These studies are consistent with the proposal that the specific deamidations at the subunit interface cause significant structural changes which lead to degradation of the protein.

Covalent modification of the active site Glu165 of triosephosphate isomerase (TPI) (EC 5.3.1.1) with the substrate analogue 3-chloroacetol phosphate (CAP) induces conformational changes similar to those observed during catalysis. We have introduced CAP into the active sites of TPI from yeast, chicken, pig, and rabbit, and assessed the effect of this modification on the structural integrity of the protein. CAP binding accelerated the specific deamidation of Asn71 in mammalian TPI. Transverse urea gradient gel electrophoretic analysis showed that the CAP-TPI dimer dissociates more readily than the native dimer. Hybrids composed of one CAP-modified subunit and one native subunit exhibited intermediate stability. The deamidated enzyme was more susceptible to proteases and denaturing conditions. Subtilisin cleaved the rabbit enzyme primarily at the Thr139-Glu140 bond. The resulting peptides remained noncovalently attached, and the enzyme retained catalytic activity. The data provide further evidence of the interactions between the catalytic center and the subunit interface and that the specific deamidation destabilizes the enzyme initiating its degradation. The enhancement of deamidation upon binding of substrate and catalysis suggest that molecular wear and tear may be involved in regulating proteolytic turnover of the enzyme.