title: Functional properties of two mutants of human glucose 6-phosphate dehydrogenase, R393G and R393H, corresponding to the clinical variants G6PD Wisconsin and Nashville.
abstract: Two severe Class I human glucose-6-phosphate dehydrogenase (G6PD, EC1.1.1.49) mutations, G6PD(Wisconsin) (nt1177 C-->G, R393G) and G6PD(Nashville) (nt1178 G-->A, R393H), affect the same codon, altering a residue in the dimer interface close to the "structural" NADP+ site. These mutations are predicted to influence interaction with the bound "structural" NADP+, long supposed to be crucial for enzyme stability. Recombinant proteins corresponding to these mutants have been constructed, expressed and purified to homogeneity. Steady-state kinetic parameters of the mutant enzymes were comparable to those of normal human G6PD, indicating that the mutations do not alter catalytic efficiency drastically. However, investigations of thermostability, urea denaturation, protease digestion, and hydrophobic exposure demonstrated that G6PD R393H is less stable than normal G6PD or R393G, and stability was more NADP+-dependent. Apoenzymes were prepared by removal of "structural" NADP+. Again the G6PD(Nashville) protein was markedly less stable, and its dissociation constant for "structural" NADP+ is approximately 500 nM, about 10 times higher than values for R393G (53 nM) and normal G6PD (37 nM). These results, together with structural information, suggest that the instability of the R393H protein, enhanced by the weakened binding of "structural" NADP+, is the likely cause of the severe clinical manifestation observed for G6PD(Nashville). They do not, however, explain the basis of disease in the case of G6PD(Wisconsin).
authors.affiliation: Department of Biochemistry, The University of Hong Kong, Hong Kong SAR.
authors.forename: Xiao-Tao
authors.initials: XT
authors.lastname: Wang
authors.forename: Veronica M S
authors.initials: VM
authors.lastname: Lam
authors.forename: Paul C
authors.initials: PC
authors.lastname: Engel
mesh_terms: D004795:Enzyme Stability; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D009249:NADP; D014508:Urea; C095433:glucose-6-phosphate dehydrogenase Wisconsin; D005954:Glucosephosphate Dehydrogenase; D014357:Trypsin