RESUMEN
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.
Asunto(s)
Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/metabolismo , NADP/metabolismo , Sitios de Unión , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Glucosafosfato Deshidrogenasa/genética , Deficiencia de Glucosafosfato Deshidrogenasa/enzimología , Humanos , Cinética , Modelos Moleculares , Peso Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica , Desnaturalización Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ácido Tricloroacético/químicaRESUMEN
Two severe Class I human glucose-6-phosphate dehydrogenase (G6PD, EC1.1.1.49) mutations, G6PD(Wisconsin) (nt1177 C-->G, R393G) and G6PD(Nashville) (nt1178 G-->A, R393H), affect the same codon, altering a residue in the dimer interface close to the "structural" NADP+ site. These mutations are predicted to influence interaction with the bound "structural" NADP+, long supposed to be crucial for enzyme stability. Recombinant proteins corresponding to these mutants have been constructed, expressed and purified to homogeneity. Steady-state kinetic parameters of the mutant enzymes were comparable to those of normal human G6PD, indicating that the mutations do not alter catalytic efficiency drastically. However, investigations of thermostability, urea denaturation, protease digestion, and hydrophobic exposure demonstrated that G6PD R393H is less stable than normal G6PD or R393G, and stability was more NADP+-dependent. Apoenzymes were prepared by removal of "structural" NADP+. Again the G6PD(Nashville) protein was markedly less stable, and its dissociation constant for "structural" NADP+ is approximately 500 nM, about 10 times higher than values for R393G (53 nM) and normal G6PD (37 nM). These results, together with structural information, suggest that the instability of the R393H protein, enhanced by the weakened binding of "structural" NADP+, is the likely cause of the severe clinical manifestation observed for G6PD(Nashville). They do not, however, explain the basis of disease in the case of G6PD(Wisconsin).
Asunto(s)
Glucosafosfato Deshidrogenasa/metabolismo , Proteínas Mutantes/metabolismo , Estabilidad de Enzimas , Calor , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , NADP/metabolismo , Desnaturalización Proteica/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Tripsina/metabolismo , Urea/farmacologíaRESUMEN
Clones overexpressing clinical glucose 6-phosphate dehydrogenase (G6PD) mutants Union (c.1360C>T/p.Arg454Cys) and Andalus (c.1361G>A/p.Arg454His), have been constructed. These abolish a salt bridge between Arg454 and Asp 286. One mutant is reportedly a Class II clinical variant and the other a Class I. Kinetic studies of the purified proteins reveal that, for both mutants, kcat is about 10-fold decreased, thus giving a 90% decrease in the WHO assay, and also presumably under physiological conditions. In contrast with unfavourable changes in Vmax for both mutants, Km values for both G6P and NADP+ are decreased approximately 5-fold. Measurements with alternative substrates confirm that G6PD Union, like the wild-type enzyme, follows a rapid-equilibrium random-order mechanism, allowing calculation of enzyme-substrate dissociation constants from initial-rate parameters. The mutations result in several-fold tighter binding of glucose 6-phosphate to the free enzyme. Binding, however, is clearly less productive than with normal enzyme. G6PD mutations are thought to cause haemolytic anaemia by compromising enzyme stability. Both these mutants indeed show somewhat decreased thermostability. However, at 37 degrees C and with NADP+, the stability differences are only moderate. Decreased catalytic efficiency clearly contributes to the disease phenotype of these two mutants, entirely accounting for reported decrease in leukocyte G6PD levels, though not for still lower levels in erythrocytes. Neither the kinetic nor the stability effects appear to justify the different clinical classification of these mutations.
Asunto(s)
Marcadores Genéticos/genética , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/genética , Anemia Hemolítica/genética , Estabilidad de Enzimas , Eritrocitos/enzimología , Humanos , Cinética , Leucocitos/enzimología , Mutación , NADP/química , Fenotipo , Plásmidos/metabolismo , TemperaturaRESUMEN
Human glucose-6-phosphate dehydrogenase (G6PD) is NADP(+)-dependent and catalyses the first and rate-limiting step of the pentose phosphate shunt. Binary complexes of the human deletion mutant, DeltaG6PD, with glucose-6-phosphate and NADP(+) have been crystallized and their structures solved to 2.9 and 2.5 A, respectively. The structures are compared with the previously determined structure of the Canton variant of human G6PD (G6PD(Canton)) in which NADP(+) is bound at the structural site. Substrate binding in DeltaG6PD is shown to be very similar to that described previously in Leuconostoc mesenteroides G6PD. NADP(+) binding at the coenzyme site is seen to be comparable to NADP(+) binding in L. mesenteroides G6PD, although some differences arise as a result of sequence changes. The tetramer interface varies slightly among the human G6PD complexes, suggesting flexibility in the predominantly hydrophilic dimer-dimer interactions. In both complexes, Pro172 of the conserved peptide EKPxG is in the cis conformation; it is seen to be crucial for close approach of the substrate and coenzyme during the enzymatic reaction. Structural NADP(+) binds in a very similar way in the DeltaG6PD-NADP(+) complex and in G6PD(Canton), while in the substrate complex the structural NADP(+) has low occupancy and the C-terminal tail at the structural NADP(+) site is disordered. The implications of possible interaction between the structural NADP(+) and G6P are considered.
Asunto(s)
Glucosa-6-Fosfato/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , NADP/metabolismo , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Interpretación Estadística de Datos , Escherichia coli/química , Escherichia coli/genética , Glucosa-6-Fosfato/química , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/genética , Humanos , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Mutación/fisiología , NADP/química , Unión Proteica , Conformación ProteicaAsunto(s)
Compensación de Dosificación (Genética) , Enfermedades Genéticas Ligadas al Cromosoma X/epidemiología , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Hemólisis/genética , Mutación/genética , Fenotipo , Factores de Edad , Anciano , Anciano de 80 o más Años , China/epidemiología , Femenino , Componentes del Gen , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Receptores Androgénicos/genética , Análisis de Secuencia de ADNRESUMEN
Cloning and over-expression of human glucose 6-phosphate dehydrogenase (Glc6P dehydrogenase) has for the first time allowed a detailed kinetic study of a preparation that is genetically homogeneous and in which all the protein molecules are of identical age. The steady-state kinetics of the recombinant enzyme, studied by fluorimetric initial-rate measurements, gave converging linear Lineweaver-Burk plots as expected for a ternary-complex mechanism. Patterns of product and dead-end inhibition indicated that the enzyme can bind NADP+ and Glc6P separately to form binary complexes, suggesting a random-order mechanism. The Kd value for the binding of NADP+ measured by titration of protein fluorescence is 8.0 microm, close to the value of 6.8 microm calculated from the kinetic data on the assumption of a rapid-equilibrium random-order mechanism. Strong evidence for this mechanism and against either of the compulsory-order possibilities is provided by repeating the kinetic analysis with each of the natural substrates replaced in turn by structural analogues. A full kinetic analysis was carried out with deaminoNADP+ and with deoxyglucose 6-phosphate as the alternative substrates. In each case the calculated dissociation constant upon switching a substrate in a random-order mechanism (e.g. that for NADP+ upon changing the sugar phosphate) was indeed constant within experimental error as expected. The calculated rate constants for binding of the leading substrate in a compulsory-order mechanism, however, did not remain constant when the putative second substrate was changed. Previous workers, using enzyme from pooled blood, have variously proposed either compulsory-order or random-order mechanisms. Our study appears to provide unambiguous evidence for the latter pattern of substrate binding.
Asunto(s)
Glucosamina/análogos & derivados , Glucosa-6-Fosfato/análogos & derivados , Glucosafosfato Deshidrogenasa/metabolismo , Glucosamina/farmacología , Glucosa-6-Fosfato/metabolismo , Glucosa-6-Fosfato/farmacología , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Humanos , Cinética , Modelos Químicos , NADP/metabolismo , NADP/farmacología , Unión Proteica , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
G6PDdb (http://www.rubic.rdg.ac.uk/g6pd/ or http://www.bioinf.org.uk/g6pd/) is a newly created web-accessible locus-specific mutation database for the human Glucose-6-phosphate dehydrogenase (G6PD) gene. The relational database integrates up-to-date mutational and structural data from various databanks (GenBank, Protein Data Bank, etc.) with biochemically characterized variants and their associated phenotypes obtained from published literature and the Favism website. An automated analysis of the mutations likely to have a significant impact on the structure of the protein has been performed using a recently developed procedure. The database may be queried online and the full results of the analysis of the structural impact of mutations are available. The web page provides a form for submitting additional mutation data and is linked to resources such as the Favism website, OMIM, HGMD, HGVBASE, and the PDB. This database provides insights into the molecular aspects and clinical significance of G6PD deficiency for researchers and clinicians and the web page functions as a knowledge base relevant to the understanding of G6PD deficiency and its management.