Regulation of the pentose phosphate cycle

1. A search was made for mechanisms which may exert a ;fine' control of the glucose 6-phosphate dehydrogenase reaction in rat liver, the rate-limiting step of the oxidative pentose phosphate cycle. 2. The glucose 6-phosphate dehydrogenase reaction is expected to go virtually to completion because the primary product (6-phosphogluconate lactone) is rapidly hydrolysed and the equilibrium of the joint dehydrogenase and lactonase reactions is in favour of virtually complete formation of phosphogluconate. However, the reaction does not go to completion, because glucose 6-phosphate dehydrogenase is inhibited by NADPH (Neglein & Haas, 1935). 3. Measurements of the inhibition (which is competitive with NADP(+)) show that at physiological concentrations of free NADP(+) and free NADPH the enzyme is almost completely inhibited. This indicates that the regulation of the enzyme activity is a matter of de-inhibition. 4. Among over 100 cell constituents tested only GSSG and AMP counteracted the inhibition by NADPH; only GSSG was highly effective at concentrations that may be taken to occur physiologically. 5. The effect of GSSG was not due to the GSSG reductase activity of liver extracts, because under the test conditions the activity of this enzyme was very weak, and complete inhibition of the reductase by Zn(2+) did not abolish the GSSG effect. 6. Preincubation of the enzyme preparation with GSSG in the presence of Mg(2+) and NADP(+) before the addition of glucose 6-phosphate and NADPH much increased the GSSG effect. 7. Dialysis of liver extracts and purification of glucose 6-phosphate dehydrogenase abolished the GSSG effect, indicating the participation of a cofactor in the action of GSSG. 8. The cofactor removed by dialysis or purification is very unstable. The cofactor could be separated from glucose 6-phosphate dehydrogenase by ultrafiltration of liver homogenates. Some properties of the cofactor are described. 9. The hypothesis that GSSG exerts a fine control of the pentose phosphate cycle by counteracting the NADPH inhibition of glucose 6-phosphate dehydrogenase is discussed.     

Intramolecular cyclization of pentose and hexose dithioacetals

The intramolecular cyclization of O-tosyl derivatives of dithioacetals of d-ribose, d-arabinose, and d-glucose was investigated. p-Toluenesulfonylation of d-glucose diethyl dithioacetal gave 3,6-anhydro-d-glucose diethyl dithioacetal. Variously substituted 5-O-tosyl-d-glucose dibenzyl dithioacetals gave derivatives of either 2,5-anhydro-l-idose dibenzyl dithioacetal, benzyl 1,5-dithio-l-idopyranoside, or l-idose dibenzyl dithioacetal. Likewise, 4-O-tosyl-d-glucose dibenzyl dithioacetal derivatives gave benzyl 1,4-dithio-d-galactofuranoside derivatives.     

Metabolic engineering for improved microbial pentose fermentation

Global concern over the depletion of fossil fuel reserves, and the detrimental impact that combustion of these materials has on the environment, is focusing attention on initiatives to create sustainable approaches for the production and use of biofuels from various biomass substrates. The development of a low-cost, safe and eco-friendly process for the utilization of renewable resources to generate value-added products with biotechnological potential as well as robust microorganisms capable of efficient fermentation of all types of sugars are essential to underpin the economic production of biofuels from biomass feedstocks. Saccharomyces cerevisiae, the most established fermentation yeast used in large scale bioconversion strategies, does not however metabolise the pentose sugars, xylose and arabinose and bioengineering is required for introduction of efficient pentose metabolic pathways and pentose sugar transport proteins for bioconversion of these substrates. Our approach provided a basis for future experiments that may ultimately lead to the development of industrial S. cerevisiae strains engineered to express pentose metabolising proteins from thermophilic fungi living on decaying plant material and here we expand our original article and discuss the strategies implemented to improve pentose fermentation.     

SIRT2 controls the pentose phosphate switch

The most common enzyme defect in humans is glucose‐6‐phosphate dehydrogenase (G6PD) deficiency, which affects more than 400 million people. G6PD shunts glucose into the pentose phosphate pathway (PPP) to generate nucleotides and reducing potential in the form of NADPH. In this issue, Wang et al ( 2014 ) show that G6PD activity is post‐translationally regulated by SIRT2, a cytoplasmic NAD⁺‐dependent deacetylase, thereby linking NAD⁺ levels to DNA repair and oxidative defences, and identifying potential new approaches to treating this common genetic disease.     

Non-oxidative synthesis of pentose 5-phosphate from hexose 6-phosphate and triose phosphate by the L-type pentose pathway

1. Ribose 5-phosphate was non-oxidatively synthesized from glucose 6-phosphate and triose phosphate by an enzyme extract prepared from rat liver (RLEP). Analysis of the intermediates by GLC, ion-exchange chromatography and specific enzymatic analysis, revealed the presence of the following intermediates of the L-type pentose pathway: altro-heptulose 1,7-bisphosphate, arabinose 5-phosphate and D-glycero D-ido octulose 8-phosphate. 2. With either [1-14C] or [2-14C]glucose 6-phosphate as diagnostic substrates, the distribution of 14C in ribose 5-phosphate was determined. At early time intervals (0.5-8 hr), [1-14C]glucose 6-phosphate introduced 14C into C-1, C-3 and C-5 of ribose 5-phosphate, at 17 hr 14C was confined to C-1. With [2-14C]glucose 6-phosphate as substrate, 14C was confined to C-2, C-3 and C-5 of ribose 5-phosphate during early times (0.5-8 hr), while at 17 hr 14C was located in C-2. 3. The transketolase exchange reaction, [14C]ribose 5-phosphate + altro-heptulose 7-phosphate in equilibrium ribose 5-phosphate + [14C]altro-heptulose 7-phosphate, was demonstrated for the first time using purified transketolase, its activity was measured and it is proposed to play a major role in the relocation of 14C into C-3 and C-5 or ribose 5-phosphate during the prediction labelling experiments. 4. The coupled transketolase-transaldolase reactions, 2 fructose 6-phosphate in equilibrium altro-heptulose 7-phosphate + xylulose 5-phosphate and 2 altro-heptulose 7-phosphate in equilibrium fructose 6-phosphate + D-glycero D-altro octulose 8-phosphate were demonstrated with purified enzymes, but are concluded to play a minor role in the non-oxidative synthesis of pentose 5-phosphate and octulose phosphate by (RLEP). 5. The formation of gem diol and dimers of erythrose 4-phosphate is proposed to account in part for the failure to detect monomeric erythrose 4-phosphate in the carbon balance studies. 6. The equilibrium value for the pentose pathway acting by the reverse mode in vitro was measured and contrasted with the value for the pathway acting in the forward direction. The initial specific rates of the pentose pathway reactions in vitro for the reverse and forward directions are measured. 7. The study which includes carbon balance, time course changes and 14C prediction labelling experiments reports a comprehensive investigation of the mechanism of the pentose pathway acting reversibly.     

The pentose phosphate pathway and parasitic protozoa

The pentose phosphate pathway plays a crucial role in the host-parasite relationship. It maintains a pool of NADPH, which serves to protect against oxidant stress and which generates carbohydrate intermediates used in nucleotide and other biosynthetic pathways. Deficiency in the first enzyme of the pathway, glucose-6-phosphate dehydrogenase, protects human erythrocytes from infection with Plasmodium falciparum for reasons that remain obscure. Loss of the third enzyme of the pathway, 6-phosphogluconate de-hydrogenase, is toxic, suggesting this enzyme might be a target for chemotherapy. Mike Barrett here summarizes the roles of the pentose phosphate pathway in various parasitic protozoa.     

Methotrexate: pentose cycle and oxidative stress

The effect of methotrexate (MTX) and leucovorin (LCV) on pentose cycle enzymes and the activity of enzymes involved in enzyme defence mechanisms against ROS in HeLa cells, were studied. The effect of MTX was also investigated on the cellular levels of glutathione. MTX inhibited the activity of glucose-6-phosphate and 6-phosphogluconate dehydrogenases. The activities of glutathione reductase and γ-glutamylcysteine synthetase were also inhibited by the drug. No effect was observed on the activities of catalase, superoxide dismutase or transketolase. LCV had no effect on any of the enzymes studied. MTX decreased the cellular levels of glutathione (70 per cent), while the presence of LCV and glutamine did not interfere with the effect of MTX. The net results appear to show that the biological situation resulting from treatment with MTX leads to a reduction of effectiveness of the antioxidant enzyme defence system. Copyright © 1998 John Wiley & Sons, Ltd.     

The pentose phosphate pathway in Trypanosoma cruzi

The pentose phosphate pathway has been studied in Trypanosoma cruzi, Clone CL Brener. Functioning of the pathway was demonstrated in epimastigotes by measuring the evolution of (14)CO(2) from [1-(14)C] or [6-(14)C]D-glucose. Glucose consumption through the PPP increased from 9.9% to 20.4% in the presence of methylene blue, which mimics oxidative stress. All the enzymes of the PPP are present in the four major developmental stages of the parasite. Subcellular localisation experiments suggested that the PPP enzymes have a cytosolic component, predominant in most cases, although all of them also seem to have organellar localisation(s).     

The game of the pentose phosphate cycle

Sugar rearrangement in the pentose phosphate cycle for transformation of six pentoses into five hexoses is analysed by abstraction to a mathematical model consisting of the resolution of a logical mathematical game of optimization. In the model, the problem is to arrive at five boxes containing six balls each, having started with six boxes containing five balls each, where boxes simulate the sugars and balls simulate the carbons in each. This is achieved by means of transferring two or three balls from any box to any other in each step, according to transketolase and transaldolase (or aldolase) mechanisms which account for sugar interconversions in the living cell. A hypothesis of simplicity is imposed in order to arrive at the objective with the least number of steps and with the least number of balls in the intermediary boxes. A symmetrical solution is obtained, demonstrating that this is the simplest solution, which is the procedure carried out by biological systems. The same treatment is applied for sugar rearrangement in the non-oxidative phase of the Calvin cycle in photosynthesis and the analysis of the "L-type" of pentose phosphate cycle is also treated, obtaining similar solutions in both cases, which allow us to make some physiological reflections.     

Reductive pentose phosphate cycle in Nitrosocystis oceanus

Campbell, Ann E. (Woods Hole Oceanographic Institution, Woods Hole, Mass.), Johan A. Hellebust, and Stanley W. Watson. Reductive pentose phosphate cycle in Nitrosocystis oceanus. J. Bacteriol. 91:1178-1185. 1966.-Assays in cell-free extracts of Nitrosocystis oceanus, a marine chemoautotrophic bacterium, have demonstrated the presence of all of the enzymes of the reductive pentose phosphate cycle, with activities high enough to account for the normal growth rate of the cells. Studies on ribulosediphosphate carboxylase activity in these extracts showed that it is inhibited by MgCl(2) (30% at 0.01 m), MnCl(2) (70% at 0.01 m), NaCl and KCl (100% at 0.5 m, 63% at 0.2 m), and by sulfate (35% at 0.01 m); phosphate, glutathione, and ethylenediaminetetraacetic acid had no effect. The bacterial enzyme differs from the spinach enzyme with respect to its affinity for bicarbonate and its pH optimum. Whole cells were incubated with C(14)O(2), and the acid-soluble fraction was analyzed by paper chromatography and autoradiography. Phosphoglyceric acid and the sugar phosphates were the earliest labeled compounds; several amino acids and organic acids were also labeled. It is concluded that N. oceanus incorporates CO(2) primarily via the reductive pentose phosphate cycle.     

高浓度糖发酵制备乙醇

以树干毕赤酵母为发酵菌株,混合糖（木糖、葡萄糖）为发酵底物,通过培养基和培养条件的改变来确定树干毕赤酵母高糖浓度发酵时所需的条件。研究结果表明：在24h发酵周期内初始木糖质量浓度为63．0g／L较适宜;在36h发酵周期内初始木糖质量浓度为72．0g/L较适宜。24h发酵周期内,在36．0g／L木糖中添加的葡萄糖质量浓度以54．0g/L为最佳,发酵结束乙醇质量浓度达32．9g／L;36h发酵周期内,添加的葡萄糖质量浓度以72．0g／L为最佳,发酵结束乙醇质量浓度为36．9g／L。以（NH4）2SO4为N源时较适合戊糖发酵制备乙醇,（NH2）2SO4的最佳质量浓度为1．1g／L。发酵前8h摇床转速为90r／min,后16h为150r／min,乙醇质量浓度较高,可达17．5g/L。