Pentose Phosphate PathwayThree main functions:1) Supply the cell with NADPH in order to: a) provide reducing power for biosynthetic reactions. b) serve as a biochemical reductant (e.g., maintain glutathione levels). c) be utilized by the cytochrome P450 monooxygenase system. d) as the electron source for reduction of ribo- to deoxyribonucleotides for DNA synthesis.2) Convert hexoses into pentoses (which are essential components of ATP, CoA, NADP+, FAD, RNA, and DNA).3) Enable the complete oxidative degradation of pentoses by converting them into hexoses and trioses which can then enter the glycolytic pathway.
The Roles of NADH and NADPH in MetabolismThere is a fundamental distinction between NADH and NADPH inmost biochemical reactions.NADH is oxidized by the electron transport chain to generate ATP.In contrast, NADPH functions as an electron donor (i.e., a hydrideion donor) in biosynthetic reactions.Recall that in the oxidation of a substrate, the nicotinamide ring ofNADP+ accepts a hydrogen ion and two electrons, which areequivalent to a hydride ion.
Stages of the Pentose Phosphate PathwayStage 1: consists of the oxidative portion of the pathway in whichtwo oxidative reactions provide NADPH and a hexose is decarboxylatedto a pentose.Stage 2: consists of two reversible isomerization reactions.Stage 3: consists of the nonoxidative portion of the pathway in whichvia a series of interconversions of three-, four-, five-, six-, andseven-carbon sugars, excess pentoses are converted to hexosesand trioses which can enter the glycolytic pathway.
Stage 1: Three reactions constitute this stage, two of which are oxidative and generate NADPH. Stage 1 is linked to biosynthetic reactions since NADPH and a pentose are produced. Glucose 6-phosphate 6-phosphogluconate Lactonase dehydrogenase dehydrogenase 1 1 3 oxidative decarboxylationThe reactions of stage 1 can be summarized as follows:Glucose 6-phosphate + 2 NADP+ + H2O ribulose 5-phosphate + 2 NADPH + 2 H+ + CO2Thus two of the three functions of the pentose phosphate pathway areaccomplished: generation of NADPH and conversion of a hexose to apentose.
Stage 2 consists of two reversible isomerization reactions which convertribulose 5-phosphate into either ribose 5-phosphate or xylulose5-phosphate.Both are substrates for the Stage 3 reactions. Phosphopentose isomerase aldose ketose Ribulose 5-phosphate can also be isomerized to xylulose 5-phosphate via the enzyme phosphopentose epimerase.
The Stage 1 + Stage 2 reactions yield 2 NADPH and 1 ribose5-phosphate for each glucose 6-phosphate oxidized.However, cells often need NADPH reducing power more than theyneed ribose 5-phosphate for nucleotide biosynthesis.In these cases, ribose 5-phosphate is further converted intoglyceraldehyde 3-phosphate and fructose 6-phosphate by theenzymes transketolase and transaldolase.These enzymes created a reversible link between the pentosephosphate pathway and glycolysis.
Reactions of Stage 3Stage 3 consists of non-oxidative reactions which link the pentosephosphate pathway with glycolysis.This stage allows:1) excess pentoses to be converted to hexoses and trioses which canthen enter glycolysis; and2) hexoses to be converted to pentoses, thereby allowing pentoseproduction without concomitant production of NADPH.Two enzymes – transketolase and transaldolase – catalyze a series ofthree reactions which convert 3 pentoses into 2 hexoses and 1 triose.These reactions involve interconversions of 3, 4, 5, 6, and7-carbon sugars.
Glyceraldehyde 3-phosphate transketolase C 5 + C5 C 3 + C7 Fructose transaldolase 6-phosphate C7 + C3 C 4 + C6 transketolase C 5 + C4 C 3 + C6 Net: 3 C5 2 C6 + 1 C3Transketolase transfers a 2-carbon fragment.Transaldolase transfers a 3-carbon fragment.
Reaction 1 Two pentoses are required: ribose 5-phosphate and xylulose 5-phosphate. A 2 carbon fragment is transferred from the ketose to the aldose. Catalyzed by transketolase. Aldose Ketose C5 C5 C3 C7Transketolase contains tightly bound TPP as its prosthetic group.Wernicke Kosakoff Syndrome is an autosomal recessive disorder caused byan alteration in transketolase which reduces its affinity for TPP.Symptoms only develop if individual suffers from a moderate thiamine deficiency.
Reaction 2The products of Reaction 1 (i.e., glyceraldehyde 3-phosphate andsedoheptulose 7-phosphate) are the substrates for Reaction 2.A 3-carbon unit is transferred from the ketose to the aldose by theenzyme transaldolase. C7 C3 C4 C6
Transketolase is also utilized for the third reaction.A 2-carbon unit is transferred from xylulose 5-phosphate (a ketose) toerythrose 4-phosphate (an aldose). C5 C4 C3 C6 Note: the products of this reaction, glyceraldehyde 3-phosphate and fructose 6-phosphate, are both intermediates of the glycolytic pathway.
The sum of the Stage 3 reactions is:2 Xylulose 5-phosphate + ribose 5-phosphate 2 fructose 6-phosphate + glyceraldehyde 3-phosphateIf we include the Stage 2 isomerization reactions, the net reaction is:3 Ribose 5-phosphate 2 fructose 6-phosphate + glyceraldehyde 3-phosphateThe important point is that excess ribose 5-phosphate formed by thepentose phosphate pathway can be completely converted intoglycolytic intermediates.
The Rate Limiting Step of the Pentose Phosphate PathwayThe first reaction in the oxidative branch of the pentose phosphatepathway catalyzed by glucose 6-phosphate dehydrogenase, is therate limiting step under physiological conditions.NADPH is a potent competitive inhibitor of the enzyme. Thus, the ratioof NADP+/NADPH regulates the pathway.As the NADP+ level rises, the flux thru the pathway increases.Note: The nonoxidative branch of the pathway is regulated primarily bysubstrate availability.
The Flow of Glucose 6-phosphate Depends on Physiological NeedThe flow of glucose 6-phosphate depends on cellular need for NADPH,ribose 5-phosphate, and ATP. The Pentose Phosphate Pathway canoperate in 4 different modes.Mode 1: Much more ribose 5-phosphate than NADPH is required.For example, rapidly dividingcells need nucleotide precursors Bypass Stage 1, feed intofor DNA synthesis more than glycolysis instead.they need NADPH.Use glycolysis + Glycolysis 2 3 Also need:Stage 2 and Stage 3 phosphopentose isomerase transketolasereactions. transaldolase phosphopentose epimerase 1
Mode 2: The needs for NADPH and ribose 5-phosphate are balanced. Oxidative Branch – Stage 1 Phosphopentose isomeraseThe stoichiometry of mode 2 is:Glucose 6-phosphate + 2 NADP+ + H2O ribose 5-phosphate + 2 NADPH + 2 H+ + CO2
Mode 3: Much more NADPH than ribose 5-phosphate is required; glucose 6-phosphate is completely oxidized to CO2. Three sets of reactions are required under these conditions. (1) Oxidative BranchThis situation typically occursin adipose tissue where a highlevel of NADPH is required for Phosphopentosefatty acid biosynthesis. (2) isomerase (2) Gluconeogensis transketolase transaldolaseRegenerate glucose 6-phosphate (3) (2)from the pentose.The sum of these reactions is:Glucose 6-phosphate + 12 NADP+ + 7 H2O 6 CO2 + 12 NADPH + 12 H+ + Pi
Mode 4: Much more NADPH than ribose 5-phosphate is required;glucose 6-phosphate is converted into pyruvate. (1) Oxidative PathwayATP, NADH, and NADPHare generated. Phosphopentose isomeraseFive of the six carbons of transketolaseglucose 6-phosphate emerge Glycolysis transaldolaseas pyruvate. (3) (2) ATP or BiosyntheticThe stoichiometry of mode 4 is: precursors3 Glucose 6-phosphate + 6 NADP+ + 5 NAD+ + 5 Pi + 8 ADP 5 pyruvate + 3 CO2 + 6 NADPH + 5 NADH + 8 ATP + 2 H2O + 8 H+
The Percentage of Glucose Metabolized by the Pentose Phosphate Pathway Varies for Different TissuesSince, a main purpose of this pathway is to supply NADPH for reductivesyntheses, it is prominent in tissues that actively carry out the reductivesynthesis of fatty acids and/or steroids from acetyl CoA.Liver: 5-10% of glucose is metabolized by the pentose phosphate pathway.Adipose Tissue: 30 – 50%Erythrocytes: 10% (need NADPH to maintain reduced glutathione)Thyroid gland, kidney, and brain: 3 – 5%Muscle: activity is extremely low.
A radioisotopic approach can be used to assess the fraction of glucosemetabolized by the pentose phosphate pathway vs. the sum ofglycolysis, PDH, + the citric acid cycle in a given tissue.Principle: Only the C-1 position of glucose is decarboxylated by thepentose phosphate pathway (at the 6-phosphogluconate dehydrogenasestep).In contrast, the C-1 and C-6 positions are decarboxylated equally whenglucose is metabolized by the glycolytic pathway, PDH, and the citricacid cycle.This is because in these pathways, the C-1 and C-6 from glucose end upas C-3 in pyruvate following the triose phosphate isomerase step. Thus,subsequent oxidation by the citric acid cycle liberates 14CO2 equally fromthe original C-1 and C-6 of glucose.
Thus, 2 samples of a given tissue are prepared:One is incubated with glucose labeled with 14C at position C-1,whereas the other sample is incubated with glucose labeled atposition C-6. The amount of radioactive 14CO2 produced by the 2samples is then compared.For example, in liver:14CO liberated by the sample incubated in glucose-1-14C is produced 2by both the citrate acid cycle and the pentose phosphate pathway;However, less 14CO2 is liberated by the sample incubated inglucose-6-14C because the citric acid cycle is the only source of14CO . 2By comparing the amount of 14CO2 released with different tissuesthat are labeled in the C-1 versus C-6 positions, one can determinethe proportion of glucose metabolized by one pathway versus another.
Role of Glucose 6-phosphate Dehydrogenase in the Red Blood CellIn the RBC glucose serves as the primary energy source. RBC’s lackmitochondria and thus lack the enzymes of the citric acid cycle.Therefore, glucose is metabolized exclusively by the glycolyticpathway (90%) and the pentose phosphate pathway (10%).The most important function of the pentose phosphate pathway in theRBC is to maintain the tripeptide glutathione in a reduced state.Oxidized glutathione is reduced by the enzyme glutathione reductasein a reaction which utilizes NADPH:
Functions of Reduced Glutathione1) To serve as a sulfhydryl buffer that maintains the cysteine residuesof hemoglobin and other RBC proteins in the reduced state.2) To maintain the iron in hemoglobin in the ferrous (i.e., reduced)form (Fe++).3) To detoxify by reacting with hydrogen peroxides and organicperoxides. This reaction is catalyzed by glutathione peroxidase. H2O2 + 2 GSH 2 H2O + GSSGThus reduced glutathione is essential for maintaining the normalstructure of RBCs. Cells with a lowered level of this compoundare more susceptible to hemolysis.Certain drugs act as oxidants formation of toxic peroxidesoxidation of proteins distortion of the surface of RBCs in theabsence of glutathione, making them more susceptible to destruction.
Glucose 6-phosphate Dehydrogenase Deficiency•Glucose 6-phosphate dehydrogenase (G6PD) deficiency is an inherited disease characterized by hemolytic anemia caused by an inability to detoxify oxidizing agents.•Most common disease-producing enzyme abnormality in humans.•Caused by a family of over 400 point mutations in the enzyme.•Only certain mutations cause clinical symptoms.•Most affected individuals have no symptoms until they are exposed to certain drugs which act as oxidizing agents.•Drug exposure can induce a hemolytic episode which in some cases can be fatal.•Life-span of many individuals with G6PD deficiency is shortened due to complications arising from chronic hemolysis.
Genetic Variants of Glucose 6-phosphate Dehydrogenase More than 400 putative variants of the G6PD have been described. Among the properties by which the variants can be distinguished are the following: 1) enzyme activity 2) kinetic properties 3) electrophoretic mobility 4) substrate specificity 5) enzyme stability 6) pH optimum The severity of the disease typically correlates with the amount of residual enzyme activity in the patient’s RBCs.
Glucose 6-phosphate Dehydrogenase Deficiency•G6PD A- is the prototype of the moderate (class III) form of the disease.•The A- variant occurs with high frequency amongst the African-American population (gene frequency of the A- allele ~ 11%) and causes a susceptibility to drug-induced hemolytic anemia.•The enzyme encoded by the A- allele is kinetically normal, but displays a substantially reduced half-life (i.e., 13 days versus 62 days).Consequence: about 3 days after an oxidizing drug is administered to anA- individual, there is a pronounced hemolytic episode. Older RBCswhich have little functional enzyme and hence are deficient in NADPHand reduced glutathione are destroyed.After ~ 1 week recovery begins since most of the remaining (i.e., youngerRBCs), as well as newly produced RBCs, have relatively normal enzymelevels.Provides protection against the drug-induced hemolysis.
•The high frequency of the A- variant suggests that the deficiency may be advantageous under certain environmental conditions.•In fact it confers partial protection against malaria (since the causative parasite requires reduced glutathione and pentose phosphate pathway products).•Illustrates the interplay between heredity and environment.The G6PD Mediterranean variant is the prototype of a more severe(class I) deficiency.•Enzyme shows normal stability, but barely detectable activity in RBCs.•A broader spectrum of drugs causes hemolysis and the hemolysis is not as self limiting as in individuals with the A- variant.
Molecular Biology of Glucose 6-phosphate Dehydrogenase•The cloning of the G6PD gene has enabled identification of mutations that cause G6PD deficiency. All are point mutations in the coding region of the gene.•Mutations causing nonspherocytic hemolytic anemia: cluster near the the NADP+ binding site.•Mutations causing milder forms of the disease cluster near the glucose 6-phosphate binding site.
Organ Integration of Carbohydrate Metabolism Liver: essential for providing fuel (i.e., glucose) to the brain, muscle, and other peripheral organs. The liver extracts Glucose from the blood. Release glucose when needed. Glycolysis Glycogen Mainly as a source Pentose Phosphate of biosynthetic Pathway intermediates; also to make some ATP NADPH for biosynthesis;Note: pentoses, hexoses.1) If there is insufficient glucose in the blood, the liver will break down its glycogen and/or synthesize glucose via gluconeogenesis to increase blood glucose supplied to other tissues.2) Liver mainly uses α-keto acids derived from amino acid catabolism to supply its own ATP needs.
Brain: Glucose is virtually the sole fuel source for the human brain,except during times of starvation. In the resting state, the brainaccounts for 60% of glucose utilization.•Glucose is obtained either from the diet or the liver.•Brain does not contain significant stores of glycogen.•Brain does not carry out gluconeogenesis since it has no glucose 6-phosphatase.Muscle: In contrast to brain, muscle has a large store of glycogen(i.e., ~ 75% of total body glycogen).•Its major fuels are glucose, fatty acids, and ketone bodies.•Like brain, muscle lacks glucose 6-phosphatase, and so it does not carry out gluconeogenesis or export glucose.•Instead muscle retains glucose, its preferred fuel, for bursts of activity.
Metabolic Interchanges Between Muscle and Liver
•During active contraction, glycolysis >> citric acid cycle. Therefore, pyruvate is reduced to lactate which then flows to liver where it is converted into glucose (Cori Cycle).•Muscle also produces much alanine (by transamination of pyruvate). This alanine can also be converted into glucose by the liver.Adipose Tissue: Triacylglycerols stored in adipose tissue providean enormous reservoir of metabolic fuel.•A principal function of this tissue is to synthesize triacylglycerols from fatty acyl CoA derivatives and glycerol 3-phosphate.•Thus, adipose cells need to metabolize glucose via glycolysis in order to provide sufficient glycerol 3-phosphate (which originates from dihydroxyacetone phosphate) for triacylglycerol synthesis.•Also, adipose cells need to carry out some pentose phosphate pathway in order to supply sufficient NADPH for synthesis.