The E104D mutation increases the susceptibility of human triosephosphate isomerase to proteolysis. Asymmetric cleavage of the two monomers of the homodimeric enzyme.
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.
Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud, México.
Ignacio
I
De La Mora-De La Mora
Alfredo
A
Torres-Larios
Guillermo
G
Mendoza-Hernández
Sergio
S
Enriquez-Flores
Adriana
A
Castillo-Villanueva
Sara T
ST
Mendez
Itzhel
I
Garcia-Torres
Angélica
A
Torres-Arroyo
Saúl
S
Gómez-Manzo
Jaime
J
Marcial-Quino
Jesús
J
Oria-Hernández
Gabriel
G
López-Velázquez
Horacio
H
Reyes-Vivas
D019943:Amino Acid Substitution; D000746:Anemia, Hemolytic, Congenital Nonspherocytic / Q000201:enzymology / Q000235:genetics; D002239:Carbohydrate Metabolism, Inborn Errors / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D006801:Humans; D020125:Mutation, Missense; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D059748:Proteolysis; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism
D014305:Triose-Phosphate Isomerase
3-chloroacetol phosphate; 5,5′-dithio-bis (2-nitrobenzoic acid); 8-anilinonaphthalene 1-sulphonate; ANS; CAP; Conformational change; DTNB; HsTIM; Limited proteolysis; PVDF; TIM deficiency; Triosephosphate isomerase; X-ray crystallography; human triosephosphate isomerase; polyvinylidene difluoride