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Probing the role of the fully conserved Cys126 in triosephosphate isomerase by site‐specific mutagenesis – distal effects on dimer stability
Author(s) -
Samanta Moumita,
Banerjee Mousumi,
Murthy Mathur R. N.,
Balaram Hemalatha,
Balaram Padmanabhan
Publication year - 2011
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2011.08110.x
Subject(s) - triosephosphate isomerase , mutant , dimer , chemistry , site directed mutagenesis , enzyme , isomerase , active site , biochemistry , stereochemistry , wild type , biophysics , crystallography , biology , gene , organic chemistry
Cys126 is a completely conserved residue in triosephosphate isomerase that is proximal to the active site but has been ascribed no specific role in catalysis. A previous study of the C126S and C126A mutants of yeast TIM reported substantial catalytic activity for the mutant enzymes, leading to the suggestion that this residue is implicated in folding and stability [Gonzalez‐Mondragon E et al. (2004) Biochemistry 43 , 3255–3263]. We re‐examined the role of Cys126 with the Plasmodium falciparum enzyme as a model. Five mutants, C126S, C126A, C126V, C126M, and C126T, were characterized. Crystal structures of the 3‐phosphoglycolate‐bound C126S mutant and the unliganded forms of the C126S and C126A mutants were determined at a resolution of 1.7–2.1 Å. Kinetic studies revealed an approximately five‐fold drop in k cat for the C126S and C126A mutants, whereas an approximately 10‐fold drop was observed for the other three mutants. At ambient temperature, the wild‐type enzyme and all five mutants showed no concentration dependence of activity. At higher temperatures (> 40 °C), the mutants showed a significant concentration dependence, with a dramatic loss in activity below 15 μ m . The mutants also had diminished thermal stability at low concentration, as monitored by far‐UV CD. These results suggest that Cys126 contributes to the stability of the dimer interface through a network of interactions involving His95, Glu97, and Arg98, which form direct contacts across the dimer interface. Database
Structural data are available in the Protein Data Bank under the accession numbers 3PVF , 3PY2 , and 3PWA . Structured digital abstract • Tim binds to Tim by x‐ray crystallography (View interaction)