TAILIEUCHUNG - Báo cáo khoa học: Cys126 is a completely conserved residue in triosephosphate isomerase that

Cys126 is a completely conserved residue in triosephosphate isomerase that is proximal to the active site but has been ascribed no specific role in catal-ysis. 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 [Gonz-alez-Mondragon Eet al. | IFEBS Journal Probing the role of the fully conserved Cys126 in triosephosphate isomerase by site-specific mutagenesis - distal effects on dimer stability Moumita Samanta1 Mousumi Banerjee1 Mathur R. N. Murthy1 Hemalatha Balaram2 and Padmanabhan Balaram1 1 Molecular Biophysics Unit Indian Institute of Science Bangalore India 2 Molecular Biology and Genetics Unit JawaharlalNehru Centre for Advanced Scientific Research Bangalore India Keywords dimer interface dimer stability Plasmodium falciparum thermal stability triosephosphate isomerase Correspondence P. Balaram Molecular Biophysics Unit Indian Institute of Science Bangalore-560012 India Fax 91 80 2360 0535 Tel 91 80 2293 3000 E-mail pb@ Received 2 February 2011 revised 22 March 2011 accepted 28 March 2011 doi 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 A. Kinetic studies revealed an approximately five-fold drop in kcat 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 IM. The mutants

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