Kinetic properties of normal human erythrocyte glucose-6-phosphate dehydrogenase dimers

Adediran, S. A.

doi:10.1016/0300-9084(91)90006-m

Cited by 11 publications

(12 citation statements)

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“…There was no indication of any such complexities in these experiments. The pattern of converging lines confirms earlier reports that human Glc6 P dehydrogenase follows a sequential mechanism [14,15].…”

Section: Discussionsupporting

confidence: 89%

“…On the other hand, early studies of human Glc6 P dehydrogenase have left the kinetic mechanism a matter of controversy, both because of the conflicting conclusions of various investigators [11–13] and because of inherent doubts about Glc6 P dehydrogenase purified from pooled, expired blood from a genetically heterogeneous population. Adediran [14] proposed an ordered‐sequential mechanism with NADP + as the leading substrate, whereas Birke et al . [15] obtained quite different results from similar experiments.…”

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confidence: 99%

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Recombinant human glucose‐6‐phosphate dehydrogenase

Wang

Lam

et al. 2002

European Journal of Biochemistry

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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 K d value for the binding of NADP + measured by titration of protein fluorescence is 8.0 lM, close to the value of 6.8 lM 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.Keywords: glucose-6-phosphate dehydrogenase; steady-state kinetics; rapid-equilibrium random-order mechanism; alternative substrate; product inhibition.Glucose-6-phosphate dehydrogenase (EC 1.l.1.49) in humans is an X-chromosome-linked housekeeping enzyme, vital for the life of every cell. It catalyses the oxidation of D-glucose 6-phosphate to D-glucono-d-lactone 6-phosphate in the first committed step of the pentose phosphate pathway, which provides cells with pentoses and reducing power in the form of NADPH. In red blood cells, this is the only source of NADPH required to protect the cells (via glutathione [1,2] and catalase [3,4]) against hydrogen peroxide and other oxidative damage. Accordingly, numerous Glc6P dehydrogenase mutations are associated with haemolytic anaemia [5].Until recently, detailed structural information was available only for the Glc6P dehydrogenase of Leuconostoc mesenteroides [6]. Extensive kinetic analysis of the NAD + -and NADP + -linked reactions for this bacterial Glc6P dehydrogenase [7][8][9][10] suggests different mechanisms for the two coenzymes. NADPH inhibition of the NADP + -linked reaction was competitive with respect to NADP + and noncompetitive with respect to Glc6P, whereas inhibition of the NAD + -...

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Section: Discussionsupporting

confidence: 89%

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confidence: 99%

Recombinant human glucose‐6‐phosphate dehydrogenase

Wang

Lam

et al. 2002

European Journal of Biochemistry

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“…The His‐tagged enzyme was characterized as a homodimer with 60‐kDa subunits, which is a common property of bacterial and eukaryal G6PDs with subunits of 50–60 kDa. Among the bacterial G6PDs dimers and tetramers were described, whereas most of the eukaryotic homologs are tetrameric proteins (for literature see [1,2,18]).…”

Section: Discussionmentioning

confidence: 99%

Glucose-6-phosphate dehydrogenase from the hyperthermophilic bacteriumThermotoga maritima: expression of theg6pdgene and characterization of an extremely thermophilic enzyme

Hansen

Schlichting

Schönheit

2002

FEMS Microbiology Letters

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The gene (open reading frame Tm1155, g6pd) encoding glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) of the hyperthermophilic bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 95 kDa composed of 60-kDa subunits. Rate dependence (at 80 degrees C) on glucose-6-phosphate and NADP(+) followed Michaelis-Menten kinetics with apparent K(m) values of 0.15 mM and 0.03 mM, respectively; apparent V(max) values were about 20 U mg(-1). The enzyme also reduced NAD(+) (apparent K(m) 12 mM, V(max) 12 U mg(-1)). The 1000-fold higher catalytic activity (k(cat)/K(m)) with NADP(+) over NAD(+) defines the G6PD as NADP(+) specific in vivo. G6PD activity was competitively inhibited by NADPH with a K(i) value of 0.11 mM. With a temperature optimum of 92 degrees C the enzyme is the most thermoactive G6PD described.

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“…The His-tagged enzyme was characterized as a homodimer with 60-kDa subunits, which is a common property of bacterial and eukaryal G6PDs with subunits of 50^60 kDa. Among the bacterial G6PDs dimers and tetramers were described, whereas most of the eukaryotic homologs are tetrameric proteins (for literature see [1,2,18]).…”

Section: Discussionmentioning

confidence: 99%

“…G6PDs with subunits of 50^60 kDa. Among the bacterial G6PDs dimers and tetramers were described, whereas most of the eukaryotic homologs are tetrameric proteins (for literature see [1,2,18]). G6PD showed a pronounced preference for both the substrate glucose-6-phosphate and the cofactor NADP þ .…”

Section: Discussionmentioning

confidence: 99%

Glucose-6-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: expression of the g6pd gene and characterization of an extremely thermophilic enzyme

Hansen

2002

FEMS Microbiology Letters

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The gene (open reading frame Tm1155, g6pd) encoding glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) of the hyperthermophilic bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 95 kDa composed of 60-kDa subunits. Rate dependence (at 80 ‡C) on glucose-6-phosphate and NADP þ followed Michaelis^Menten kinetics with apparent K m values of 0.15 mM and 0.03 mM, respectively; apparent V max values were about 20 U mg 31 . The enzyme also reduced NAD þ (apparent K m 12 mM, V max 12 U mg 31 ). The 1000-fold higher catalytic activity (k cat /K m ) with NADP þ over NAD þ defines the G6PD as NADP þ specific in vivo. G6PD activity was competitively inhibited by NADPH with a K i value of 0.11 mM. With a temperature optimum of 92 ‡C the enzyme is the most thermoactive G6PD described.

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Kinetic properties of normal human erythrocyte glucose-6-phosphate dehydrogenase dimers

Cited by 11 publications

References 27 publications

Recombinant human glucose‐6‐phosphate dehydrogenase

Recombinant human glucose‐6‐phosphate dehydrogenase

Glucose-6-phosphate dehydrogenase from the hyperthermophilic bacteriumThermotoga maritima: expression of theg6pdgene and characterization of an extremely thermophilic enzyme

Glucose-6-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: expression of the g6pd gene and characterization of an extremely thermophilic enzyme

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