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 approximately 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 K d for structural NADP+ as 37 nM, 200-fold lower than for "catalytic" NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37 degrees C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4 degrees 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.
Wang
Xiao-Tao
XT
Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong SAR.
Chan
Ting Fai
TF
Lam
Veronica M S
VM
Engel
Paul C
PC
D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005955:Glucosephosphate Dehydrogenase Deficiency / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014238:Trichloroacetic Acid / Q000737:chemistry
D050505:Mutant Proteins; D011994:Recombinant Proteins; D009249:NADP; D014238:Trichloroacetic Acid; D005954:Glucosephosphate Dehydrogenase