The importance of NADP(+) for stability is explained by the structural NADP(+) site, which is not conserved in prokaryotes. The structure shows that point mutations causing severe deficiency predominate close to the structural NADP(+) and the dimer interface, primarily affecting the stability of the molecule. They also indicate that a stable dimer is essential to retain activity in vivo. As there is an absolute requirement for some G6PD activity, residues essential for coenzyme or substrate binding are rarely modified.

We wish to record here her important contribution to research on human G6PD.

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 ¼ 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 There have been many reports over the years that the subunits of human glucose 6-phosphate dehydrogenase (G6PD) [EC 1.1.1.49] possess a second NADP + -binding site in addition to that directly involved in catalysis (Kirkman 1962;Kirkman and Hendrickson 1962;Chung and Langdon 1963;Bonsignore et al. 1970;Yoshida 1973). The existence and possible role of this postulated ''structural'' NADP + site remained controversial, however, not least because the first solved G6PD structure, that of the homologous enzyme of Leuconostoc mesenteroides (Lm), showed no sign of any coenzyme site other than the catalytic binding site (Rowland et al. 1994). The issue was finally and unambiguously resolved, however, by solution of the corresponding structure for human G6PD (the Canton mutant) (Au et al. 2000), which clearly shows bound NADP + occupying a second site, quite distinct from the vacant catalytic NADP + site. Considerable interest now attaches to the role of this tightly bound nucleotide molecule, since the crystallographic structure reveals that a high proportion of the clinical...

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