1 Chapter 14b (Summary) Takusagawa’s note Summary of Chapter 14b 1. Gluconeogenesis - is the biosynthesis of glucose from non-carbohydrate precursors at liver and kidney (minor). - Glycogen stored in liver is only a half day supply of glucose to brain which uses only glucose as fuel. - Initially, glycolysis products (pyruvate & lactate), citric acid cycle intermediates, and carbon skeletons of most amino acids are converted to oxaloacetate. - Gluconeogenesis uses the reversed glycolysis pathway, except for 3 steps which have large positive ∆G. Those and alternated pathways are: 1. “Pyruvate + ATP → PEP + ADP” by pyruvate kinase is replaced with: - HCO3 + ATP ADP + P i GTP GDP + CO2 Pyruvate Oxaloacetate Phosphoenol pyruvate (PEP) Pyruvate carboxylase PEP carboxykinase 2. “FBP + ADP → F6P + ATP” by phosphofructokinase is replaced with: H2 O Pi Fructose-1,6-bisphosphate Fructose-6-phosphate Fructose bisphosphatase 3. “G6P + ADP → Glucose + ATP” by hexokinase is replaced with: Pi H2O Glucose-6-phosphate Glucose Glucose-6-phosphatase 2. As shown above, gluconeogenesis is accomplished by avoiding three energetically unfavorable reverse reactions and by expensing hydrolysis of 4ATP and 2GTP per glucose. Glucose + 2NAD+ + 2ADP + 2Pi → 2(Pyruvate) + 2NADH + 4H+ + 2ATP + 2H2O Glucose + 2NAD+ + 4ADP + 6Pi + 2GDP ← 2(Pyruvate) + 2NADH + 4H+ + 4ATP + 6H2O + 2GTP Overall (glycolysis & gluconeogenesis): 2ADP + 4Pi + 2GDP ↔ 2ATP + 4H2O + 2GTP 3. Pyruvate carboxylase has a biotin prosthetic group which is covalently bound to the enzyme by amide linkage between its -COO- and H3N+-(CH2)4- of Lys residue. Biotin is at the end of 14 Å long flexible arm. This is quite similar to the lipoic acid attached to Lys residue in pyruvate dehydrogenase multienzyme complex. - Biotin is a coenzyme and CO2 carrier, and is an essential human nutrient. 4. Acetyl-CoA is a powerful allosteric activator of pyruvate carboxylase since Acetyl-CoA requires oxaloacetate to continue the citric acid cycle pathway. 5. Gluconeogenesis only occurs when the citric acid cycle is inhibited by excess of ATP and/or NADH. 6. PEP in mitochondrion is transported through the specific transport protein whereas oxaloacetate is transported by malate-aspartate shuttle since oxaloacetate does not have specific transport proteins. - Oxaloacetate is reduced to malate by mitochondrial NADH. The transported malate reduces the cytosolic NAD+ to NADH and becomes oxaloacetate. The produced NADH is utilized at 1,3-BPG → GAP. 7. Regulation of gluconeogenesis - Glycolysis and gluconeogenesis pathways are reciprocally regulated at three independent pathways: 1. Glucose ↔ G6P [HK / glucose-6-phosphatase] 2. F6P ↔ FBP [PFK / FBPase] 3. PEP ↔ Pyruvate [PK / pyruvate carboxylase-PEPCK] - Dominant mechanisms are: 1. Allosteric regulations - Fructose-2,6-bisphosphate (F2,6P) is a potent activator of glycolysis (activate PFK, inhibit FBPase). - F2,6P is synthesized and hydrolyzed by two enzyme activities (PFK-2 and FBPase-2). F6P → F2,6P → F6P 1 2 Chapter 14b (Summary) Takusagawa’s note PFK-2 FBPase-2 2. cAMP-dependent covalent modifications (initiated by glucagon and other hormonal activity). - Phosphorylation of liver {PFK-2/FBPase-2} by cAPK inhibits PFK-2 and activates FBPase-2 activities. - Thus, phosphorylation of {PFK-2/FBPase-2} by cAPK decreases PFK activity and increases FBPase activity, i.e., decreases glycolysis and increase gluconeogenesis in liver. Note: Heart {PFK-2/FBPase-2} responds oppositely. However, gluconeogenesis takes place in liver. - Liver pyruvate kinase (PK) is inhibited allosterically by Ala (precursor of pyruvate), and inactivated by phosphorylation. 8. Cori cycle --- In muscle {glucose → pyruvate → lactate} via blood to liver {lactate → pyruvate → glucose} via blood to muscle {glucose → pyruvate → lactate}. This cycle process looks ATP/GTP consuming “futile cycle”, but does not take place in the same cell. 9. Glyoxylate pathway (Only plants) - Acetyl-CoA cannot enter gluconeogenesis pathway in animals, but in plants, Acetyl-CoA can enter gluconeogenesis via glyoxylate pathway. Glyoxylate + acetyl-CoA → malate, catalyzed by malate synthase. Glyoxysome Mitochondrion Aspartate Oxaloacetate Oxaloacetate Aspartate C.A.C + Acetyl-CoA Succinate Isocitrate Glyoxylate Glucose + Acetyl-CoA Gluconeogenesis Oxaloacetate Malate Malate Citric acid cycle: Acetyl-CoA → 2CO2 + 2NADH + GTP → Oxidative phosphorylation → ATP - Glyoxylate pathway: Acetyl-CoA → Glyoxylate → Gluconeogenesis → Glucose 10. Biosysnthesis of oligosaccharides and glycoproteins - Glycosyl donors are nucleotide-sugars such as UDP-glucose, and oligosaccharide synthses are catalyzed by glycosyl transferases. - Glycoproteins are classified into three groups. 1. N-linked oligosaccharides. - The oligosaccharides are constructed on dolichol in the lumen of the rough endoplasmic reticulum. - A common 14-residue core oligosaccharide is transfered to the newly synthesized polypeptide at an Asn. 2. O-linked oligosaccharides. - A monosaccharide is attached to Ser or Thr with a-O-glycosidic bond mostly, and then additional oligosaccharides are attached one by one on a completed polypeptide chain in Golgi apparatus. 3. GPI-linked proteins. - GPI core is synthesized in ER from phosphatidylinositol, UDP-GlcNAc and Dolichol-P-mannose. 11. Pentose phosphate pathway - produces two important biomolecules (NADPH and ribose-5-phosphate (R5P)) from G6P. - ~30% of glucose oxidation in liver occurs via the pentose phosphate pathway. - Although NADH and NADPH are chemically similar, those are not metabolically interchangeable. - NADPH is the reducing power currency in cell. For example, H2O2 and R-O-O-H produced in cell are very toxic compounds, and thus must be reduced to H2O and R-OH, respectively. Glutathione (GSH) is mainly used as reducing agent in cell. The oxidized glutathione (GSSG) is subsequently regenerated by NADPH. - In cell, [NAD+]/[NADH] ≈ 1000 vs. [NADP+]/[NADPH] ≈ 0.01. What does this mean? - Pentose phosphate pathway has three stages: 1. 3G6P + 6NADP+ + 3H2O → 3Ru5P + 6NADPH + 6H+ + 3CO2 2. 3Ru5P → R5P + 2Xu5P 3. R5P + 2Xu5P → 2F6P + GAP - 2 3 Chapter 14b (Summary) - - Takusagawa’s note Overall reaction: 3G6P +6NADP + 3H2O → 2F6P + GAP + 6NADPH + 6H+ + 3CO2 Pentoses cannot directly enter the glycolysis, gluconeogenesis or pentose phosphate pathway, thus they are converted to hexoses (F6P) and triose (GAP). Two enzymes are involved. 1. Transketolase that adds and/or subtracts C2 units: C5 + C5 ↔ C3 + C7 2. Transaldolase that adds and/or subtracts C3 units: C3 + C7 ↔ C6 + C4 Transketolase has a TPP co-factor which carries the C2-unit from one sugar to another sugar. Transaldolase forms a Schiff base with the substrates, which carries the C3-unit from one sugar to another sugar. One G6P can produce 12 NADPH by 6 cycles of pentose phosphate pathway and gluconeogenesis. Pentose phosphate pathway (NADPH production) is controlled by the rate of glucose-6-phosphate dehydrogenase (first committed step with ∆G = -17.6 kJ/mol in liver). + 3