The E104D mutation increases the susceptibility of human triosephosphate isomerase to proteolysis. Asymmetric cleavage of the two monomers of the homodimeric enzyme

• Published in: Biochimica et biophysica acta Vol. 1834; no. 12; pp. 2702 - 2711
• Main Authors: De La Mora-De La Mora, Ignacio, Torres-Larios, Alfredo, Mendoza-Hernández, Guillermo, Enriquez-Flores, Sergio, Castillo-Villanueva, Adriana, Mendez, Sara T., Garcia-Torres, Itzhel, Torres-Arroyo, Angélica, Gómez-Manzo, Saúl, Marcial-Quino, Jaime, Oria-Hernández, Jesús, López-Velázquez, Gabriel, Reyes-Vivas, Horacio
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2013
• Subjects: Amino Acid Substitution; Anemia, Hemolytic, Congenital Nonspherocytic - enzymology; Anemia, Hemolytic, Congenital Nonspherocytic - genetics; Carbohydrate Metabolism, Inborn Errors - enzymology; Carbohydrate Metabolism, Inborn Errors - genetics; cardiomyopathy; Conformational change; death; enzyme inactivation; Enzyme Stability - genetics; Humans; Limited proteolysis; mutants; mutation; Mutation, Missense; pathogenesis; peptidase K; Protein Multimerization; Protein Structure, Quaternary; Proteolysis; TIM deficiency; triose-phosphate isomerase; Triose-Phosphate Isomerase - chemistry; Triose-Phosphate Isomerase - deficiency; Triose-Phosphate Isomerase - genetics; Triose-Phosphate Isomerase - metabolism; Triosephosphate isomerase; X-ray crystallography; X-ray crystallography; HsTIM; CAP; DTNB; TIM deficiency; ANS; Triosephosphate isomerase; Limited proteolysis; PVDF; Conformational change; 8-anilinonaphthalene 1-sulphonate; polyvinylidene difluoride; 3-chloroacetol phosphate; human triosephosphate isomerase; 5,5′-dithio-bis (2-nitrobenzoic acid)
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2013.08.012

The deficiency of human triosephosphate isomerase (HsTIM) generates neurological alterations, cardiomyopathy and premature death. The mutation E104D is the most frequent cause of the disease. Although the wild type and mutant exhibit similar kinetic parameters, it has been shown that the E104D substitution induces perturbation of an interfacial water network that, in turn, reduces the association constant between subunits promoting enzyme inactivation. To gain further insight into the effects of the mutation on the structure, stability and function of the enzyme, we measured the sensitivity of recombinant E104D mutant and wild type HsTIM to limited proteolysis. The mutation increases the susceptibility to proteolysis as consequence of the loss of rigidity of its overall 3-D structure. Unexpectedly, it was observed that proteolysis of wild type HsTIM generated two different stable nicked dimers. One was formed in relatively short times of incubation with proteinase K; as shown by spectrometric and crystallographic data, it corresponded to a dimer containing a nicked monomer and an intact monomer. The formation of the other nicked species requires relatively long incubation times with proteinase K and corresponds to a dimer with two clipped subunits. The first species retains 50% of the original activity, whereas the second species is inactive. Collectively, we found that the E104D mutant is highly susceptible to proteolysis, which in all likelihood contributes to the pathogenesis of enzymopathy. In addition, the proteolysis data on wild type HsTIM illustrate an asymmetric conduct of the two monomers. •The E104D mutant of HsTIM is highly susceptible to limited proteolysis.•The mutant raises its susceptibility by the loss of rigidity of the 3D structure.•Such behavior most likely contributes to the pathogenesis of enzymopathy.•Proteolysis data of wild type show an asymmetric conduct of its two subunits.