Lecture 6 - Other Pathways of Carbohydrate Metabolism PDF

Other pathways of carbohydrate metabolism Prof. Dr. Gerhard Grüber Nanyang Technological University School of Biological Sciences [email protected]  ATP yield per molecule of glucose at each stage of cellular respiration CYTOSOL...

Other pathways of carbohydrate metabolism Prof. Dr. Gerhard Grüber Nanyang Technological University School of Biological Sciences [email protected]  ATP yield per molecule of glucose at each stage of cellular respiration CYTOSOL Electron shuttles MITOCHONDRION span membrane 2 NADH or 2 FADH2 2 NADH 2 NADH 6 NADH 2 FADH2 GLYCOLYSIS PYRUVATE OXIDATION OXIDATIVE CITRIC PHOSPHORYLATION ACID Glucose 2 Pyruvate 2 Acetyl CoA CYCLE (Electron transport and chemiosmosis) + 2 ATP + 2 ATP + about 26 or 28 ATP Maximum per glucose: About 30 or 32 ATP © 2017 Pearson Education, Ltd  The fate of carbon in the TCA cycle The oxidation–reduction enzymes and coenzymes are shown in magenta. Entry of the two carbons of acetyl-CoA into the TCA cycle are indicated with the green box. The carbons released as CO2 are shown with yellow boxes. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Glyoxylate cycle: An anabolic variant of the citric acid cycle of depredate lipids,a a acetyl-coi L pold C4 The glyoxylate cycle allows to metabolize two-carbon substrates, such as acetate by clearage of eliminating the CO2-producing reactions and 66 molecula enhancing the net production of four-carbon dicarboxyl acids. Co2 is not generated in this Glyoxylate cycle bypasses the decarboxylation steps of the TCA cycle to ensure pathway net synthesis of oxalacetate. The citrate synthase, aconitase, and malate dehydrogenase of the glyoxylate cycle are isozymes of the citric acid cycle enzymes; isocitrate lyase and malate synthase are unique to the glyoxylate cycle. Notice that two acetyl groups (pink) enter the cycle and four carbons leave as succinate (blue). Mathews, van Holde, Ahern: Biochemistry 3rd edition Raven, Johnson, Losos, Mason & Singer: Biology 8th edition  Relationship between glyoxylate- and TCA cycle The reactions of the glyoxylate cycle (in glyoxysomes) proceed simultaneously with, and mesh with, those of the citric acid cycle (in mitochondria), as intermediates pass between these compartments. The glyoxylate cycle results in the net conversion of two acetyl-Co to succinate in the glyoxysome, which can be converted to malate in the mitochondrion for use in gluconeogensis. The conversion of succinate to oxaloacetate is catalyzed by citric acid cycle enzymes. by malate - aspartate shuttle , enter into cutoplasm & in to) happens cytosol Voet, Voet: BIOCHEMISTRY 3rd edition  Summary: The glyoxylate cycle ⚫ Acetyl-CoA condenses with oxaloacetate to give citrate and then isomerizes to isocitrate as in the TCA cycle. ⚫ Instead of being decarboxylated, isocitrate is cleaved by isocitrate lyase into succinate and glyoxylate. ⚫ Glyoxylate condenses with another acetyl-CoA to form malate in a reaction catalyzed by malate synthase. ⚫ Malate is oxidized to oxaloacetate as in the TCA cycle. ⚫ Succinate passes into the mitochondrial matrix and enters the TCA cycle to form malate. Malate has two options: (1) stays in TCA cycle to produce energy, or (2) passes into the cytosol and converted by gluconeogenesis to Fructose-6-P, the precursor of sucrose.  The glyoxylate cycle Why has plant invented glyoxylate cycle? ⚫ Plants store lipids in their seeds, to be used as energy source and biosynthetic precursors during germination, before photosynthesis takes over. ⚫ Acetyl-CoA, derived from fatty acid oxidation, is used through gluconeogenesis to produce glucose and then sucrose and many other metabolites. ⚫ In plant seedlings, sucrose provides much of the chemical energy needed for initial growth. ⚫ Why do plant seeds store fuel as lipids rather than carbohydrates? For easier dispersion, the seeds should be lighter in weight. Lipids, as fuel, is 2-fold lighter than carbohydrates: 1 g of lipids produce twice as much calories than by 1 g of carbohydrates. & of much as they are a higher reduced state compared to carbohydrates  Overview of carbohydrate metabolism anotherform of decarboxylate pathway After glucose is transported into cells, it is phosphorylated by a hexokinase to form glucose 6- Cl phosphate. Glucose 6-phosphate can then enter 25 several metabolic pathways. The three that are 20 is eliminated) common to all cell types are glycolysis, the pentose phosphate pathway, and glycogen synthesis (Fig. right). Important. The phosphate group in glucose 6- phosphate (G6-P) is completely ionized at physiological pH, giving it an overall negative charge. Major pathways of glucose metabolism. Since the plasma membrane is impermeable to charged molecules, G6-P cannot enter into the cells from the blood stream. In tissues, fructose and galactose are converted to intermediates of glucose metabolism. Thus, the fate of these sugars parallels that of glucose (Fig. right). Overview of fructose and galactose metabolism. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Link of glycolycis and the pentose phosphate pathway The pentose phosphate pathway generates NADPH for reactions that require reducing equivalents (electrons) or ribose 5-P for nucleotide biosynthesis. Glucose 6-P is a decarboxylat substrate for both the pentose phosphate pathway and glycolysis. The five-carbon 25 sugar intermediates of the pentose phosphate pathway are reversibly interconverted to intermediates of glycolysis. The portion of glycolysis that is not part of the pentose phosphate pathway is shown in magenta. The enzymes of the pentose phosphate pathway are particularly abundant in the cytoplasm of liver and adipose cells. Remember, the NADPH-dependent fatty acid synthesis is located in the cytoplasm. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  The pentose phosphate pathway The first enzyme of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase, oxidizes the trl] - Gop aldehyde at carbon 1 and reduces NADP+ to NADPH. The gluconolactone that is formed is rapidly hydrolyzed to 6-phosphogluconate, a sugar acid with a carboxylic acid group at carbon 1. The next oxidation step releases this carboxyl group as CO2, with the electrons being transferred to NADP+. This reaction is mechanistically very similar to the one catalyzed by isocitrate dehydrogenase in the TCA cycle. Thus, 2 moles of NADPH per mole of glucose 6-P are formed from this portion of the pathway. I 1 02 & Rusp The carbon skeleton of R5P and the atoms derived from it are drawn in magenta and those from Xu5P are drawn in green. Voet, Voet: BIOCHEMISTRY 3rd edition  The pentose phosphate pathway The nonoxidative portion of the pentose phosphate pathway consists of a series of rearrangement and transfer reactions that first convert ribulose 5-P to ribose 5-P and xylulose 5-phosphate (xylulose 5-P), and then the ribose epimerizat 5-P and xylulose 5-P are converted to ~ intermediates of the glycolytic pathway. The enzymes involved are epimerase, isomerase, transketolase, and transaldolase. Interchange of groups on a single carbon is an epimerization, and interchange of groups b e t w e e n c a r b o n s i s a n isomerizaton. Voet, Voet: BIOCHEMISTRY 3rd edition  fixes = molecule toe catalytic & centre & new attack occurs The mechanism of the TPP-dependent releasing t r a n s k e t o l a s e r e a c t i o n. G3p The transketolase enzyme acts at step 6 and 8 of the pentose phosphate pathway by catalyzing the transfer of two-carbon units. In these reactions the donor molecule is a ketose (D-Xylulose-5-P) and the recipient an aldose (Ribose-5-P or Erythrose-4P). The enzyme is TPP-dependent, and the mechanism involves abstraction of the Cy ~ 2C unit acidic thiazole proton, attack by the carbanion at the carbonyl carbon of the ketose phosphate t substrate, expulsion of the glyceraldehyde-3-P product, and the transfer of the two-carbon unit. C7 The role of TPP here is, thus, very similar to its C2 role in the oxidative decarboxylation of pyruvate a n d α - k e t o g l u t a r a t e. Two reactions in the pentose phosphate pathway use transketolase. In the first, the two-carbon keto fragment from xylulose 5-P is transferred to ribose 5-P to form sedoheptulose 7-phosphate (sedoheptulose 7-P); and in the other, a two- carbon keto fragment (usually derived from xylulose 5-P) is transferred to erythrose 4- phosphate (erythrose 4-P) to form fructose 6-P. Garett & Grisham: Biochemistry 4th edition  The pentose phosphate pathway Transaldolase transfers a three-carbon keto fragment from sedoheptulose 7-P to glyceraldehyde 3-P to form erythrose 4-P and fructose 6-P (Fig. right). The aldol cleavage occurs between the two hydroxyl carbons adjacent to the keto group (on carbons 3 and 4 of the sugar). This reaction is similar to the aldolase reaction in glycolysis, and the enzyme uses an active amino group from the side chain of lysine to catalyze the reaction. C can enter in glycolysis Voet, Voet: BIOCHEMISTRY 3rd edition  A balanced sequence of reactions in the pentose phosphate pathway The net result of the metabolism of 3 mol of ribulose 5-P in the pentose phosphate pathway is the formation of 2 mol of fructose 6-P and 1 mol of glyceraldehyde 3-P, which then continue through the glycolytic pathway with the production of NADH, ATP, and pyruvate. Because the pentose phosphate pathway begins with glucose 6-P and feeds back into the glycolytic pathway, it is sometimes called the hexose monophosphate (HMP) shunt (a shunt or a pathway for glucose 6-P). The reaction sequence starting from glucose 6-P, involving both the oxidative and nonoxidative phases of the pathway, is shown in the right Figure. The entry of glucose 6-P into the pentose phosphate pathway is controlled by the cellular concentration of NADPH. NADPH is a strong product inhibitor of glucose-6-phosphate dehydrogenase, the first enzyme of the pathway. As NADPH is oxidized in other pathways, the product inhibition of glucose-6-phosphate dehydrogenase is relieved, and the rate of the enzyme is accelerated to produce more NADPH. In the liver, the synthesis of fatty acids from glucose is a major route of NADPH reoxidation. The synthesis of liver glucose-6-phosphate dehydrogenase, like the key enzymes of glycolysis and fatty acid synthesis, is induced by the increased insulin:glucagon ratio after a high-carbohydrate meal. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Fructose conversion to C3-molecules Glycolysis Fructose is metabolized by conversion to glyceraldehyde 3-phosphate (glyceraldehyde 3-P) and dihydroxyacetone phosphate, which are intermediates of glycolysis (Fig. right). The steps parallel those of glycolysis. The first step in the metabolism of fructose, as with glucose, is phosphorylation. Fructokinase phosphorylates fructose in the 1-position. Fructokinase has a high Vmax and rapidly phosphorylates fructose as it enters the cell. The fructose 1-phosphate (fructose 1-P) formed is not an intermediate of glycolysis but rather is cleaved by aldolase B to dihydroxyacetone phosphate (an intermediate of glycolysis) and glyceraldehyde. Glyceraldehyde is then phosphorylated to glyceraldehyde 3-P by triose kinase. Dihydroxyacetone phosphate and glyceraldehyde 3-P are intermediates of the glycolytic pathway and can proceed through it to a) pyruvate, b) the tricarboxylic acid (TCA) cycle, and c) fatty acid synthesis. Alternatively, these ~ intermediates can also be converted to glucose by d) acetyl-cA gluconeogenesis. In other words, the fate of fructose parallels that of glucose. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Role of the Pentose Phosphate Pathway in generation of NADPH USH reduce Hror , one of pos [o) [U) 20) [H) PPP In general, the oxidative phase of the pentose phosphate pathway is the major source of NADPH in cells. NADPH provides the reducing equivalents for biosynthetic reactions and for oxidation–reduction reactions involved in protection against the toxicity of reactive oxygen species (ROS). The glutathione-mediated defense against oxidative stress is common to all cell types (including red blood cells), and the requirement for NADPH to maintain levels of reduced glutathione (GSH) probably accounts for the universal distribution of the pentose phosphate pathway among different types of cells. NADPH is also used for anabolic pathways, such as fatty acid synthesis, cholesterol synthesis, and fatty acid chain elongation. It is the source of reducing equivalents for cytochrome P450 hydroxylation of aromatic compounds, steroids, alcohols, and drugs. The highest concentrations of glucose-6-phosphate dehydrogenase are found in phagocytic cells where NADPH oxidase uses NADPH to form superoxide from molecular oxygen. The superoxide then generates hydrogen peroxide, which kills the microorganisms taken up by the phagocytic cells. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition Coffee break  Overview of glucose metabolism Mathews, van Holde, Ahern: Biochemistry 3rd edition  The need of gluconeogenesis perform gluconeogenesis The brain, CNS, erythrocytes, testes, renal medulla and embryonic tissues require glucose from blood as their sole or major fuel source. Human brain alone requires 120 g of glucose each day. Total glucose content in all our body fluids is about 20 g, and the total glycogen storage at any given time is about 190 g. So that supply of readily available glucose is not worth of one day if one has to maintain. The steady-state blood glucose level is about 5 mM. Therefore, glucose must recycle. Synthesis and use of glucose Under fasting or starvation, gluconeogenesis becomes in the human body. The liver and kidneys account for about 90% and even more important. Gluconeogenesis provides over 60% 10% of the body’s gluconeogenesis, of total glucose used over the first day of fasting. respectively. Mathews, van Holde, Ahern: Biochemistry 3rd edition  Glycolysis and gluconeogenesis in the liver Gluconeogenesis, which occurs primarily in the liver, is the pathway for the synthesis of glucose from compounds other than carbohydrates. In humans, the major precursors of glucose are lactate, glycerol, and amino acids, particularly alanine. Except for three key sequences, the reactions of gluconeogenesis are reversals of the steps of glycolysis. The sequences of gluconeogenesis that do not use enzymes of glycolysis involve the irreversible, regulated steps of glycolysis. These three sequences are the conversion of (1) pyruvate to phosphoenolpyruvate (PEP), (2) fructose 1,6-bisphosphate to fructose 6-phosphate, and (3) glucose 6-phosphate to glucose. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Precursors for Gluconeogenesis The precursors for gluconeogenesis are amino acids (particularly alanine), lactate, and glycerol. Heavy red arrows indicate steps that differ from those of glycolysis. & comes from fats catabolism catabolism of peptides, proteins C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Metabolism of gluconeogenic precursors to process ~ to] A. Conversion of lactate to pyruvate. B. Conversion of alanine to pyruvate. In this reaction, alanine aminotransferase transfers the [H] amino group of alanine to α- ketoglutarate (α-kg) to form glutamate. The coenzyme for this reaction, pyridoxal phosphate, accepts and donates the amino group. C. Conversion of glycerol to dihydroxyacetone phosphate (DHAP). in equ C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Precursors for Gluconeogenesis (C4) + 2 (C3) C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Conversion of pyruvate to oxalacetate (OAA) GO' = -2.1 KJ/mol Mechanism of the Pyruvate carboxylase: The enzyme operates by a two-step mechanism, starting with an ATP-dependent carboxylation of the cofactor to give N-carboxybiotin. This activated derivative then transfers the carboxyl directly to pyruvate. Pyruvate decarboxylase uses biotin as a cofactor, which is linked to the -amino group of a lysine residue, forming a 14 Å long arm, which swings to transfer the carboxyl. Y cofactor Biotin as a CO2 carrier in the pyruvate carboxylate Mathews, van Holde, Ahern: Biochemistry 3rd edition Voet, Voet: BIOCHEMISTRY 3rd edition  Gluconeogenesis requires metabolic transport Pyruvate carboxylation is a [O] compartmentalized reaction. TH) Pyruvate is converted to oxalacetate in the mitochondria. Because oxalacetate cannot be transported across the mitochondrial membrane, it must be reduced to malate, transported to the cytosol, and then oxidized back to oxalacetate before gluconeogenesis can continue. or by aspartate shuttle where , oxaloacetate undergoes amino trsf to become aspartate , transported to cytosol & amino test to form aspartate back oxaloacetate Transport of PEP and Oxalacetate Note,that Oxalacetate can be converted to Malate or Aspartate for further transport. Garett & Grisham: Biochemistry 4th edition Voet, Voet: BIOCHEMISTRY 3rd edition  Conversion of Pyruvate to Phosphoenolpyruvate decarboxylated carbonyl Nu undergo attack to substrate triphosphate Lenergy donor Note, that a) GTP is an energy donor in this reaction and b) that the same CO2, that was fixed by the pyruvate decarboxylase is released in this reaction, so that no net fixation of CO2 occurs. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Reaction of the Fructose-1,6-biphosphatase The hydolysis of fructose-1,6- biphosphate to fructose-6-phosphate condenses is an exergonic reaction under standard conditions. The fructose- 1,6-bisphosphatase is an allosteric e n z y me. Ci t ra t e s t i m m u l a t e s biphosphatase activity, but fructose- & 2,6-bisphosphatase is a potent allosteria center allosteric inhibitor. AMP also Fructose-1,6-bisphosphatase from pig with fructose-6- inhibits the biphosphatase, the phosphate, AMP, amd Mg2+. inhibition by AMP is enhanced by fructose-2,6-biphosphatase. Garett & Grisham: Biochemistry 4th edition  Reaction of the Glucose-6-Phosphatase histidine O inorganic phosphate irreversible Nu attack histidine process - regenerated phosphonistidine intermediate The glucose-6-phosphatase reaction involves a phosphorylated enzyme intermediate, phosphohistidine. The G for the glucose-6-phosphatase reaction in liver is -5.1 kJ/mol. bring in The glucose-6-phosphatase is present in the membranes of the ER of liver and kidney cells but absence in muscle and brain. For this reason, gluconeogenesis is not carried out in muscle and brain. The glucose-6-phosphatase system includes the phosphatase itself and three transport proteins, T1, T2 and T3. & GLUT 2 Garett & Grisham: Biochemistry 4th edition  Coupling with hydrolysis of ATP and GTP drives gluconeogenesis The net reaction of gluconeogensis: 2 pyruvate + 2 NADH + 2 H+ + 4 ATP + 2 GTP + 6 H2O → glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi The net free energy change for this conversion is -37.7 KJ/mol. The consumption of a total of six nucleoside triphosphates drives the process forward. If glycolysis were merely reversed to achieve the net synthesis of glucose from pyruvate, the net reaction would be 2 pyruvate + 2 ATP + 2 NADH + 2 H+ + H2O → glucose + 2 ADP + 2 Pi + 2 NAD+ Hydrolysis of four additional high-energy phosphate bonds makes gluconeogenesis thermodynamically favourable. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Major controling mechanisms affecting glycolysis and gluconeogenesis citrate + Garett & Grisham: Biochemistry 4th edition  Sources of Blood Glucose Immediately after a meal, dietary carbohydrates serve as the major source of blood glucose. As blood glucose levels return to the fasting range within 2 breaks down glycogen hours after a meal, glycogenolysis is in liver synthesis of glucose non-carbohydrate stimulated and begins to supply glucose to the blood. Subsequently, glucose is - - from sources also produced by gluconeogenesis. During a 12-hour fast, glycogenolysis is the major source of blood glucose. Thus, it is the major pathway by which glucose is produced in the basal state (after a 12- hour fast). However, by approximately 16 hours of fasting, glycogenolysis and gluconeogenesis contribute equally to the maintenance of blood glucose. By 30 hours after a meal, liver glycogen stores are substantially depleted. Subsequently, gluconeogenesis is the primary source of blood glucose. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Quiz 1. What are the two enzymes enabling plants two metabolize acetate and where are these enzymes located? ⚫ Isocitrate is cleaved by isocitrate lyase into succinate and glyoxylate. ⚫ Glyoxylate condenses with another acetyl-CoA to form malate in a reaction catalyzed by malate synthase. ⚫ Both enzymes are located in the glycosomes. 2. Why can gluconeogenesis not be merely the reversal of glycolysis ? Gluconeogenesis cannot be merely the reversal of glycolysis as glycolysis is exergonic with a GO’ of about -74 KJ/mol. If gluconeogenesis were merely the reverse, it would be a strongly endergonic process and could not occur spontaneously.  Quiz 3. What is the prosthetic group of pyruvate carboxylase ? Biotin is the prosthetic group. 4. Which amino acid residue is involved in the glucose-6-phosphatase reaction, which catalyzes glucose-6-P into glucose? The glucose-6-phosphatase reaction includes a phosphorylated enzyme intermediate, phosphohistidine. 5. Call two important regulators of the gluconeogensis? Acetyl-CoA and fructose-2,6-biphosphate  Quiz 6. What are the two phases of the pentose phosphate pathway and what are the two important products of this pathway? ⚫ The pathway consists of an oxidative and a nonoxidative phase. ⚫ The two important products are NADPH and ribose 5-phosphate. NADPH provides the reducing power for biosynthesis of fatty acids and steroids. Ri-5P is needed for nucleic acid biosynthesis, which occurs at high rate in growing and regenerating tissues, and in tumors. 7. What is the prosthetic group of the transketolases in the nonoxidative phase of the pentose phosphate pathway? The prosthetic group of transketolases of the nonoxidative phase of the pentose phosphate pathway is thiamine pyrophosphate-dependent (TPP). The enzymes transfer a two-carbon fragment. Thank you!