What is the role of the second structural NADP+-binding site in human glucose 6-phosphate dehydrogenase?

Human glucose 6- phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/ subunit of acid- extractable `` structural'' NADP+ and no NADPH. This tightly bound NADP+ was reduced by G6P, presumably following migration to the catalytic site. Gel- filtration yielded apoenzyme, devoid of bound NADP+ but, surprisingly, still fully active. Mr of the main component of `` stripped'' enzyme by gel filtration was; 100,000, suggesting a dimeric apoenzyme ( subunit Mr 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate Mr of 150 kDa. Fluorescence titration of the stripped enzyme gave the Kd for structural NADP+ as 37 nM, 200- fold lower than for `` catalytic'' NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37 C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4 C was partially reversed at room temperature, especially with added NADP+. Apoenzyme was immediately active, without any visible lag, in rapid- reaction studies. Human G6PD thus forms active dimer without structural NADP+. Apparently, the true role of the second, tightly bound NADP+ is to secure long- term stability. This fits the clinical pattern, G6PD deficiency affecting the long- lived non- nucleate erythrocyte. The Kd values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively ( seven- and 13- fold elevated), matching the structural prediction of weakened structural NADP+ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of Kd constant for structural NADP+ will now allow quantitative assessment of this defect in clinical G6PD mutations.