Abstract
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in the world. More than 160 mutations causing the disease have been identified, but only 10% of these variants have been studied at biochemical and biophysical levels. In this study we report on the functional and structural characterization of three naturally occurring variants corresponding to different classes of disease severity: Class I G6PD Durham, Class II G6PD Santa Maria, and Class III G6PD A+. The results showed that the G6PD Durham (severe deficiency), and the G6PD Santa Maria and A+ (less severe deficiency) (Class I, II and III, respectively) affect the catalytic efficiency of these enzymes, are more sensitive to temperature denaturing, and affect the stability of the overall protein when compared to the wild type WT-G6PD. In the variants, the exposure of more and buried hydrophobic pockets was induced and monitored with 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence, directly affecting the compaction of structure at different levels and probably reducing the stability of the protein. The degree of functional and structural perturbation by each variant correlates with the clinical severity reported in different patients.
Highlights
The deficiency of glucose-6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49) has been recognized as the most common enzymopathy, because it affects near 400 million people worldwide [1]
The mutants have a higher affinity for substrate glucose 6-phosphate (G6P) with respect to the WT-G6PD enzyme (Table 2). This decrease in Km value was observed in the mutant Union (Class I) [22], Andalus (Class II) [22] and Valladolid (Class II) [7]. This change seems favorable, at saturation levels of G6P, these variant enzymes have a diminished catalytic efficiency; we suggest that the higher affinity for G6P substrate is a compensatory mechanism due to the decrease of the catalytic efficiency
We have reported on the structural and functional characterization of three naturally occurring variants corresponding to different classes of disease severity
Summary
The deficiency of glucose-6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49) has been recognized as the most common enzymopathy, because it affects near 400 million people worldwide [1]. The G6PD is an X-linked house-keeping cytosolic enzyme [2] and participates in the first step of the pentose phosphate pathway, catalyzing the conversion of glucose 6-phosphate (G6P) to 6-phosphogluconolactone with the concomitant production of one molecule of nicotinamide adenine dinucleotidInet. The TGhe6PdeDficeiennzcyyomf gelupcloasye-s6-pahfouspnhdaatemdeehnytdarlogroenleasein(Ge6rPyDt)h(rEoCc1y.1t.e1s.4,9d) huaes bteoenthreecomgnaizinedtenance of haemoglobasinthaenmdosot tchomermponroetneziynmsoipnathay,rbeedcuaucseedit satfafetcetstnheaatr 4d0e0pmeinlldiosn poenoptlhe ewogrlludwtaitdheio[1n].eThaentioxidant system [3].G6PD is an X-linked house-keeping cytosolic enzyme [2] and participates in the first step of. Inththeisswuobrks,tiwtue rteiopnortothfeacdonesntriuncetiofno,r guanine nucleotidecGl(o6nPntiD)n,g(lA,oecxaÑpterdeGsisn)iodanitfafpenrdoensditetpitaoairnltesd7o1ffu3tnh(ceetipxornooantel ia7nn,)d,anrsedtrsuwuceltutcironamgl spitaunrdetiethhseeoscfehtwharintehegtenheeowrfeccllyoinmsiicbnailenavtnaotrihaaunrtmgsiaonnfine amino acid residuWeT2-G368P(DKeÑnzyRm)e.[8T,h9e] C(FlaisgsuI,reG61P)D, wDhuirchhamisinnveoalvretsothtehesusbtsrtiutucttiuonraolfNadAenDinPe+fosritgeu, aanninde is related with severneucclelointiidcea(lntm) (Aan→ifeGs)taatipoonssitioans71h3e(mexoolnit7y),creasunlatienmg iinathaencdhancgherofnliycsinneotnosaprghineirnoecaymtiicnohemolityc anaemia. Schematic representation of human G6PD enzyme (PDB code 2BH9) [17]. TThheeyyiieellddffrroommtthheeppuurriiffiiccaattiioonn ooff tthheeCCllaassssIIGG66PPDDDDuurrhhaammvvaarriiaannttwwaassaapppprrooxxiimmaatteellyy 2211%%,, wwhhiilleetthheeCCllaassssIIII GG66PPDD SSaannttaa--MMaarriiaa aanndd CCllaassss IIIIII GG66PPDD AA++vvaarriiaannttssggaavveeyyiieellddssooff3311%%aanndd4433%%,, rreessppeeccttiivveellyy. AAsseexxppeecctteedd,,tthheeppuurriiffiiccaattiioonn yyiieelldd ooff tthhee GG66PPDD AA++iissvveerryyssiimmiillaarrttootthhaattoobbttaaiinneeddffoorr tthhee WWTT--GG66PPDD eennzzyymmee. The specific activity was measured under standard conditions as described in Materials and Methods
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