Banerjee, Mousumi and Balaram, Hemalatha and Joshi, NV and Balaram, P (2011) Engineered dimer interface mutants of triosephosphate isomerase: the role of inter-subunit interactions in enzyme function and stability. In: Protein Engineering Design and Selection, 24 (5). pp. 463-472.
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The role of inter-subunit interactions in maintaining optimal catalytic activity in triosephosphate isomerase (TIM) has been probed, using the Plasmodium falciparum enzyme as a model. Examination of subunit interface contacts in the crystal structures suggests that residue 75 (Thr, conserved) and residue 13 (Cys, variable) make the largest number of inter-subunit contacts. The mutants Cys13Asp (C13D) and Cys13Glu (C13E) have been constructed and display significant reduction in catalytic activity when compared with wild-type (WT) enzyme (similar to 7.4-fold decrease in k(cat) for the C13D and similar to 3.3-fold for the C13E mutants). Analytical gel filtration demonstrates that the C13D mutant dissociates at concentrations < 1.25 mu M, whereas the WT and the C13E enzymes retain the dimeric structure. The order of stability of the mutants in the presence of chemical denaturants, like urea and guanidium chloride, is WT > Cys13Glu > Cys13Asp. Irreversible thermal precipitation temperatures follow the same order as well. Modeling studies establish that the Cys13Asp mutation is likely to cause a significantly greater structural perturbation than Cys13Glu. Analysis of sequence and structural data for TIMs from diverse sources suggests that residues 13 and 82 form a pair of proximal sites, in which a limited number of residue pairs may be accommodated.
|Item Type:||Journal Article|
|Additional Information:||Copyright of this article belongs to Oxford University Press.|
|Keywords:||dimer stability;inter-subunit interactions;Plasmodium falciparum;subunit interface;triosephosphate isomerase|
|Department/Centre:||Division of Biological Sciences > Centre for Ecological Sciences
Division of Biological Sciences > Molecular Biophysics Unit
|Date Deposited:||20 May 2011 06:54|
|Last Modified:||20 May 2011 06:54|
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