2004
DOI: 10.1021/bi036077s
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Conserved Cysteine 126 in Triosephosphate Isomerase Is Required Not for Enzymatic Activity but for Proper Folding and Stability

Abstract: In triosephosphate isomerase, Cys126 is a conserved residue located close to the catalytic glutamate, Glu165. Although it has been mentioned that Cys126 and other nearby residues are required to maintain the active site geometry optimal for catalysis, no evidence supporting this idea has been reported to date. In this work, we studied the catalytic and stability properties of mutants C126A and C126S of Saccharomyces cerevisiae TIM (wtTIM). None of these amino acid replacements induced significant changes in th… Show more

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Cited by 61 publications
(73 citation statements)
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“…A 0.1 μM VC1 solution (2 mL) was titrated with successive volumes of 0.02 μM and 0.2 μM QUIN. We adjusted the titration data set according to previous authors [48,49], assuming that the fluorescence intensities of the emitting species (free VC1 and VC1–QUIN complex) are additive, according to Equation 1, where x is the total concentration of inhibitor in the cell, Et represents the total concentration of VC1, K d is the dissociation equilibrium constant ( K d = 1/ K b ) and a denotes the asymptotic value to which Y tends at high x values. Y = 1-F/F t , were F is the overall fluorescence intensity after each addition of QUIN and F t is the fluorescence of free VC1 at the corresponding concentration, respectively.…”
Section: Methodsmentioning
confidence: 99%
“…A 0.1 μM VC1 solution (2 mL) was titrated with successive volumes of 0.02 μM and 0.2 μM QUIN. We adjusted the titration data set according to previous authors [48,49], assuming that the fluorescence intensities of the emitting species (free VC1 and VC1–QUIN complex) are additive, according to Equation 1, where x is the total concentration of inhibitor in the cell, Et represents the total concentration of VC1, K d is the dissociation equilibrium constant ( K d = 1/ K b ) and a denotes the asymptotic value to which Y tends at high x values. Y = 1-F/F t , were F is the overall fluorescence intensity after each addition of QUIN and F t is the fluorescence of free VC1 at the corresponding concentration, respectively.…”
Section: Methodsmentioning
confidence: 99%
“…We, therefore, developed a consensus approach for locating them in TIM-barrel proteins (11). As thermodynamic and kinetic experiments show (12,13), SRs identified by our algorithm have a significant role in the stabilization of protein structures. Thus, we believe that our definition of SRs can be a useful tool for scientists in the exploration of structural stability of proteins.…”
Section: Introductionmentioning
confidence: 82%
“…The removal of free cysteine residue is a well-known strategy to enhance protein thermostability due to the involvement of free cysteine in chemical modification and thiol-disulfide interexchange [14,[24][25][26]. However, the mechanism of such effect has seldom Figure 3.…”
Section: Discussionmentioning
confidence: 99%