Thermodynamic and Kinetic Destabilization of Triosephosphate Isomerase Resulting from the Mutation of Conserved and Non-conserved Cysteines

Ma.      Eugenia Cruces-Angeles; Nallely      Cabrera; Ruy      Perez-Montfort; Cesar      A. Reyes-Lopez; Andres      Hernandez-Arana

Abstract

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.

Keywords: protein stability, protein evolution, cysteine mutation, invariant residues, unfolding-refolding kinetics, kinetic stability, TIM, Ytim, DGAP, CD, PCR, NdeI, SDS-PAGE, JASCOprotein stability, protein evolution, cysteine mutation, invariant residues, unfolding-refolding kinetics, kinetic stability, TIM, Ytim, DGAP, CD, PCR, NdeI, SDS-PAGE, JASCO