title: Clinical mutants of human glucose 6-phosphate dehydrogenase: impairment of NADP(+) binding affects both folding and stability.
abstract: 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(Wisconsin) (R393G) and G6PD(Nashville) (R393H), and G6PD(Fukaya) (G488S) and G6PD(Campinas) (G488V), in which the mutations are in the vicinity of the "structural" NADP(+) site, showed elevated K(d) 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 degrees C. The refolding yields of the mutants exhibited strong NADP(+)-dependence and ranged from 1.5% to 59.4% with 1000 microM 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 cause instability of these clinical mutant enzymes in the fully folded state.
authors.affiliation: School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Xiao-Tao
authors.initials: XT
authors.lastname: Wang
authors.forename: Paul C
authors.initials: PC
authors.lastname: Engel
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D004795:Enzyme Stability; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007231:Infant, Newborn; D008958:Models, Molecular; D009154:Mutation; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding
chemical_list: D009249:NADP; D005954:Glucosephosphate Dehydrogenase