Functional properties of two mutants of human glucose 6-phosphate dehydrogenase, R393G and R393H, corresponding to the clinical variants G6PD Wisconsin and Nashville

AbstractTwo severe Class I human glucose-6-phosphate dehydrogenase (G6PD, EC1.1.1.49) mutations, G6PDWisconsin (nt1177 C→G, R393G) and G6PDNashville (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 G6PDNashville protein was markedly less stable, and its dissociation constant for “structural” NADP+ is ∼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 G6PDNashville. They do not, however, explain the basis of disease in the case of G6PDWisconsin