Purification and kinetic characterization of hexokinase and glucose-6-phosphate dehydrogenase fromSchizosaccharomyces pombe

1998 ◽  
Vol 76 (1) ◽  
pp. 107-113 ◽  
Author(s):  
C Stan Tsai ◽  
Q Chen
Keyword(s):  
Pentose Phosphate Pathway ◽  
Ternary Complex ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Pentose Phosphate ◽  
Oxidative Pentose Phosphate Pathway ◽  
Phosphoryl Group ◽  
Maximal Velocity ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Yeast Hexokinase

Hexokinase and D-glucose-6-phosphate dehydrogenase (G6PDH) from Schizosaccharomyces pombe have been purified 250-fold by an identical three-step. Both enzymes are dimeric with a molecular mass of 88 kDa for the kinase and 112 kDa for the dehydrogenase. Steady-state kinetic studies were performed on hexokinase and G6PDH, which form the glucose phosphate branch of the oxidative pentose phosphate pathway of S. pombe (fission yeast). Hexokinase promotes Mg2+-activated phosphorylation of D-glucose by the equilibrium random Bi Bi mechanism with formation of the abortive enzyme-ADP-glucose complex. ADP inhibits the kinase competitively versus ATP and noncompetitively versus D-glucose. The Mg2+activation of hexokinase is associated with an increase in the maximal velocity by its interaction with the ternary complex to facilitate the transfer of the phosphoryl group. G6PDH catalyzes NADP+-linked oxidation of D-glucose-6-phosphate by the ordered Bi Bi mechanism with NADP+as the leading reactant. High NADP+concentration inhibits the dehydrogenase by forming the dead-end ternary complex. In addition, G6PDH is also subjected to product inhibition by NADPH and noncompetitive inhibition by A(G)TP. Thus, the oxidative pentose phosphate pathway in S. pombe may be regulated via inhibition of hexokinase by ADP in conjunction with inhibition of G6PDH by NADPH and ATP.Key words: yeast hexokinase, glucose-6-phosphate dehydrogenase.

II. Lokalisation von Enzymen des reduktiven und dem oxydativen Pentosephosphat-Zyklus in den Chloroplasten und Permeabilität der Chloroplasten-Membran gegenüber Metaboliten

1967 ◽  
Vol 22 (11) ◽  
pp. 1200-1215 ◽  
Author(s):  
U. Heber ◽  
U. W. Hallier ◽  
M. A. Hudson ◽  
B. von der Groeben ◽  
R. Ernst ◽  
...  
Keyword(s):  
Pentose Phosphate Pathway ◽  
Enzyme Activities ◽  
Intracellular Localization ◽  
Pentose Phosphate ◽  
Oxidative Pentose Phosphate Pathway ◽  
Amino Acid Synthesis ◽  
Phosphogluconate Dehydrogenase ◽  
Chloroplast Membrane ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Isolated Chloroplasts

1. The interrelationship of metabolic activities in chloroplasts and cytoplasm of leaf cells of spinach, sugar beet and Elodea has been investigated. Different methods have been adopted to study the intracellular localization of enzymes and the flow of phosphorylated intermediates across the chloroplast membrane. The flow of substrates was investigated by determining the rates of the conversion of substrates added to aqueously isolated chloroplasts, prior to and after destruction of the outer chloroplast membrane. The observed differences yielded information as to whether a substrate could traverse the chloroplast membrane.Two methods mere used to investigate the localization of enzymes :a) The percentage distribution of photosynthetic and respiratory enzymes in chloroplasts and cytoplasm was calculated from data on enzyme activities in non-aqueous cell fractions.b) Low levels of enzymes in chloroplasts in the presence of high cytoplasmatic levels were detected by assaying enzyme activities in preparations of aqueously isolated chloroplasts prior to and after ultrasonic destruction of the outer chloroplast membrane.2. If chloroplasts are isolated in aqueous sucrose buffer, their outer membranes act as an efficient barrier against the penetration of NADP, RuDP, GAP and, in some but not all experiments, of FMP and GMP. PGA, DHAP and, probably to a lesser extent, aspartate, ɑ-ketoglutarate, oxaloacetate and FDP can traverse this membrane. Chloroplast membranes are significantly altered when isolated in NaCI-buffer systems and do not correspond to the in vivo situation.3. The conversion of Ri-5-P to RuDP occurs exclusively or nearly exclusively in the chloroplasts indicating that phosphoribulokinase and/or ribosephosphate isomerase are located only there.4. The conversion of Ri-5-P to GAP and SuMP, which is catalyzed by the enzymes ribosephosphate isomerase, xylulosephosphate epimerase and transketolase, proceeds likewise only or at least predominantly in the chloroplasts and not, or only to a small extent, in the cytoplasm.5. The major parts of glucose-6-phosphate dehydrogenase and of 6-phosphogluconate dehydrogenase reside in the cytoplasm. However, a small, but significant, level of these enzymes is to be found also in the chloroplasts. Hexokinase and transaldolase are also present there. Pyruvate kinase and phosphofructokinase appear to be absent from chloroplasts.6. Since, with the presence of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, hexokinase, transaldolase and enzymes of the Calvin cycle, the enzymic machinery of the oxidative pentose phosphate pathway is complete in the chloroplasts, the results suggest that chloroplasts are engaged in the oxidative decomposition of carbohydrates.7. In the dark the oxidative pentose phosphate pathway requires the control of NADPH formation and the transfer of hydrogen across the chloroplast membrane.8. The available data on the intracellular localization of enzymes and on the kinetics of the distribution of labelled intermediates show that the photosynthetic carbon cycle operates exclusively within the chloroplasts. There is nothing to suggest that enzymes of chloroplasts and cytoplasm cooperate in the cyclic regeneration of the carbon acceptor molecule. However, the existence of phosphorylated transport metabolites suggests that secondary reactions of photosynthesis such as sucrose and amino acid synthesis, which proceed, at least in part, outside the chloroplasts, are directly linked with chloroplastic reactions by activated (phosphorylated) intermediates.

The effect of soaking injury in bean seeds on aspects of the oxidative pentose phosphate pathway in embryonic axes

1992 ◽  
Vol 2 (1) ◽  
pp. 33-39 ◽  
Author(s):  
Johan C. Pretorius ◽  
J. G. Chris Small
Keyword(s):  
Pentose Phosphate Pathway ◽  
Causative Factor ◽  
Pentose Phosphate ◽  
Oxidative Pentose Phosphate Pathway ◽  
Embryonic Axes ◽  
Phosphogluconate Dehydrogenase ◽  
Saturated Water ◽  
Radicle Emergence ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Seed Coats

AbstractSubmerging Phaseolus vulgaris cv. Top Crop seeds in air-saturated water for 16 h markedly depresses subsequent germination. This is termed soaking injury. Soaking injury does not occur in seeds soaked in CO2-saturated water. Previous studies have shown that soaking injury can be alleviated by drying seeds or removing seed coats. Submergence therefore leads to a situation in bean seeds which is similar to secondary dormancy.As with dormant seeds, C6/C1 ratios of embryonic axes of seeds soaked in air-saturated water remained high (0.8–1.0) during and after soaking. This was paralleled by low activities of glucose-6-phosphate dehydrogenase (EC.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44). In axes of seeds soaked in CO2-saturated water and in unsoaked seeds C6/C1 ratios declined steadily during soaking/imbibition and reached values of around 0.3 after germination. Slight increases ofglucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities occurred in the pre-germination phase. This was followed by a massive increase after radicle emergence. Synthesis of the plastid isoenzymes was a post-germinative event.It appears that soaking injury depresses protein synthesis. Lack of oxidative pentose phosphate pathway activity appears to be a causative factor in soaking injury.

Regulation of D-glucose-6-phosphate dehydrogenase from Schizosaccharomyces pombe

1998 ◽  
Vol 76 (4) ◽  
pp. 645-648
Author(s):  
C Stan Tsai ◽  
Q Chen
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Pentose Phosphate Pathway ◽  
Pentose Phosphate ◽  
Oxidative Pentose Phosphate Pathway ◽  
Hill Coefficient ◽  
Negative Cooperativity ◽  
Hill Coefficients ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
The Hill

D-Glucose-6-phosphate dehydrogenase is a regulatory enzyme of the oxidative pentose phosphate pathway in Schizasaccharomyces pombe. The enzyme is subject to negative cooperative regulation by D-glucose-6-phosphate as characterized by the Hill coefficient of 0.68 ± 0.04. D-Glyceraldehyde-3-phosphate and D-ribulose-5-phosphate rectify the negative cooperativity as evidenced from a change in the Hill coefficients to 0.98 ± 0.05 and 1.02 ± 0.05, respectively. These pentose phosphate pathway intermediates also inhibit the enzyme competitively with respect to D-glucose-6-phosphate. Thus, D-glucose-6-phosphate dehydrogenase provides an avenue for regulating the partitioning of D-glucose between the redundant branches of the oxidative phosphate pathway in S. pombe.Key words: regulation, yeast, glucose-6-phosphate dehydrogenase.

scholarly journals Targeting oxidative pentose phosphate pathway prevents recurrence in mutant Kras colorectal carcinomas

PLoS Biology ◽  
2019 ◽  
Vol 17 (8) ◽  
pp. e3000425 ◽  
Author(s):  
WenChao Gao ◽  
YuTing Xu ◽  
Tao Chen ◽  
ZunGuo Du ◽  
XiuJuan Liu ◽  
...  
Keyword(s):  
Pentose Phosphate Pathway ◽  
Pentose Phosphate ◽  
Oxidative Pentose Phosphate Pathway ◽  
Colorectal Carcinomas
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