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

Abstract

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...

Functional and Biochemical Analysis of Glucose-6-Phosphate Dehydrogenase (G6PD) Variants: Elucidating the Molecular Basis of G6PD Deficiency

“…The N-terminal domain (amino acids contains the β-α-β dinucleotide binding site with a Rossmann type folding (amino acids 38-44), where the active site of the enzyme that binds β-D-glucose-6-phosphate (G6P) and NADP + is located; and the second binding site consists of an antiparallel nine-strand sheet located near the interface region of the protein known as the "structural NADP + binding site" (Figure 1) [7,8]. Interestingly, the second structural NADP + binding site is present only in higher organisms and is involved in the dimerization and the stability of the enzyme [6,9]. It is noteworthy that mutations occurring near the structural binding site of NADP + decrease the stability of the enzyme, causing severe phenotypes such as CNSHA [10].…”

Intracellular NADPH Levels Affect the Oligomeric State of the Glucose 6-Phosphate Dehydrogenase

“…Crystal structure and biochemical analyses of human G6PDH have shown that each subunit of this enzyme binds, well separated from the catalytic coenzyme binding site, a structural molecule of NADP ϩ , between the dimer interface and the C terminus (3,19,39). We propose that the replacement of NADP ϩ with NADPH causes a conformational change and/or a variation in the net charge/oligomeric state of K. lactis G6PDH that leads to the appearance of a new G6PDH band with faster migrating properties.…”

Updates on Glucose 6-Phosphate Dehydrogenase (G6PD): From Prokaryotes to Human

“…However, NADP + which is required to aid folding and monomer hybridization is bound at the structural site and not involved in the catalysis of the enzyme [16,103,104]. The auxiliary cofactor helps to maintain the stability and integrity of the enzyme when it is in its active form [105]. Generally, the structural site of NADP + is found and conserved in higher organisms [98].…”

“…The side chain of Lys-148 is less tightly bound in subunit A than in subunit B as a result of the different conformations of the two proline residues (A149 and B149) in the two subunits. This leads to a tighter binding of phosphate in subunit B since only one phosphate binds in subunit B; although limited knowledge is provided on this [104,105]. A theoretical reason might be that the specificity of the site of phosphate is greater if Pro-149 exist in a Trans in subunit B (Figure 3).…”