TY - JOUR
T1 - Thermodynamic and kinetic destabilization of triosephosphate isomerase resulting from the mutation of conserved and non-conserved cysteines
AU - Cruces-Ángeles, Ma Eugenia
AU - Cabrera, Nallely
AU - Pérez-Montfort, Ruy
AU - Reyes-López, César A.
AU - Hernández-Arana, Andrés
PY - 2011/12
Y1 - 2011/12
N2 - Several variants of Saccharomyces cerevisiae triosephosphate isomerase (yTIM) were studied to determine how mutations of conserved and non-conserved Cys residues affect the enzyme. Wild-type yTIM has two buried free cysteines: Cys 41 (non-conserved) and the invariant Cys 126. Single-site mutants, containing substitutions of these cysteines with Ala, Val, or Ser (the three most conservative changes for a buried Cys, according to substitution matrices), were examined for stability and enzymatic activity. Neither of the Cys residues was found to be essential for enzyme catalysis. Determination of the global stability of the mutants indicated that, regardless of which Cys was substituted, individual Cys-Ala and Cys-Val mutations, as well as the C41S substitution, all decrease the unfolding free energy of the dimeric protein by less than 23 kJ mol-1 (at 37 °C, pH 7.4), as compared to the wild-type enzyme. In contrast, a substantially larger destabilization (37 kJ mol-1) was found in the C126S mutant. These results suggest that, with the exception of C126S, all of these mutations can be regarded as neutral (i.e., mutations that do not impair the reproductive success of the organism). Accordingly, Cys 126 has remained invariant across evolution because its neutral substitutions by Ala or Val would require a highly unlikely, concerted double mutation at any of the Cys codons. Furthermore, detrimental effects to a cell expressing the C126S TIM mutant more likely arise from the high unfolding rate of this enzyme.
AB - Several variants of Saccharomyces cerevisiae triosephosphate isomerase (yTIM) were studied to determine how mutations of conserved and non-conserved Cys residues affect the enzyme. Wild-type yTIM has two buried free cysteines: Cys 41 (non-conserved) and the invariant Cys 126. Single-site mutants, containing substitutions of these cysteines with Ala, Val, or Ser (the three most conservative changes for a buried Cys, according to substitution matrices), were examined for stability and enzymatic activity. Neither of the Cys residues was found to be essential for enzyme catalysis. Determination of the global stability of the mutants indicated that, regardless of which Cys was substituted, individual Cys-Ala and Cys-Val mutations, as well as the C41S substitution, all decrease the unfolding free energy of the dimeric protein by less than 23 kJ mol-1 (at 37 °C, pH 7.4), as compared to the wild-type enzyme. In contrast, a substantially larger destabilization (37 kJ mol-1) was found in the C126S mutant. These results suggest that, with the exception of C126S, all of these mutations can be regarded as neutral (i.e., mutations that do not impair the reproductive success of the organism). Accordingly, Cys 126 has remained invariant across evolution because its neutral substitutions by Ala or Val would require a highly unlikely, concerted double mutation at any of the Cys codons. Furthermore, detrimental effects to a cell expressing the C126S TIM mutant more likely arise from the high unfolding rate of this enzyme.
KW - Cysteine mutation
KW - Invariant residues
KW - Kinetic stability
KW - Protein evolution
KW - Protein stability
KW - Unfolding-refolding kinetics
UR - http://www.scopus.com/inward/record.url?scp=80054878255&partnerID=8YFLogxK
U2 - 10.2174/092986611797642715
DO - 10.2174/092986611797642715
M3 - Artículo
SN - 0929-8665
VL - 18
SP - 1290
EP - 1298
JO - Protein and Peptide Letters
JF - Protein and Peptide Letters
IS - 12
ER -