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Article Dans Une Revue Biochimica et Biophysica Acta - Molecular Basis of Disease Année : 2009

Clinical mutants of human glucose 6-phosphate dehydrogenase: Impairment of NADP binding affects both folding and stability

Xiao-Tao Wang
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Paul C. Engel
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Résumé

Human glucose 6-phosphate dehydrogenase (G6PD) has both the “catalytic” NADP site and a “structural” NADP site where a number of severe G6PD deficiency mutations are located. Two pairs of G6PD clinical mutants, G6PD (R393G) and G6PD (R393H), and G6PD (G488S) and G6PD (G488V), in which the mutations are in the vicinity of the “structural” NADP site, showed elevated values of the “structural” NADP, ranging from 53 nM to 500 nM compared with 37 nM for the wild-type enzyme. These recombinant enzymes were denatured by Gdn-HCl and refolded by rapid dilution in the presence of L-Arg, NADP and DTT at 25 C. The refolding yields of the mutants exhibited strong NADP- dependence and ranged from 1.5% to 59.4% with 1000 µM NADP, in all cases lower than the figure of 72% for the wild-type enzyme. These mutant enzymes also displayed decreased thermostability and high susceptibility to chymotrypsin digestion, in good agreement with their corresponding melting temperatures in CD experiments. Taken together, the results support the view that impaired binding of “structural” NADP can hinder folding as well as causing instability of these clinical mutant enzymes in the fully folded state.
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Dates et versions

hal-00506511 , version 1 (28-07-2010)

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Xiao-Tao Wang, Paul C. Engel. Clinical mutants of human glucose 6-phosphate dehydrogenase: Impairment of NADP binding affects both folding and stability. Biochimica et Biophysica Acta - Molecular Basis of Disease, 2009, 1792 (8), pp.804. ⟨10.1016/j.bbadis.2009.05.003⟩. ⟨hal-00506511⟩

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