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

¹ Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong SAR
² School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland

(RECEIVED March 15, 2008; FINAL REVISION April 22, 2008; ACCEPTED April 23, 2008)

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. M_r of the main component of "stripped" enzyme by gel filtration was 100,000, suggesting a dimeric apoenzyme (subunit M_r = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate M_r of 150 kDa. Fluorescence titration of the stripped enzyme gave the K _d 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 K _d 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 K _d constant for structural NADP⁺ will now allow quantitative assessment of this defect in clinical G6PD mutations.

Keywords: glucose 6-phosphate dehydrogenase; apoenzyme; "structural" NADP⁺ ; cofactor removal; enzyme stability; dissociation constant; G6PD deficiency

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