Chapter 6 Carbohydrate Metabolism Jia-Qing Zhang 张嘉晴 Biochemistry department Medical college Jinan university Mar. 2007

What’s metabolism?

Metabolism….. What is Life? What are the properties of life? Movement Reproduction of one’s kid Metabolism

Carbohydrate metabolism Protein metabolism Lipid metabolism

metabolism Carbohydrate metabolism Metabolism of lipid Catabolism of protein

Carbohydrate Metabolism

Section 1 Introduction Carbohydrates are the major source of carbon atoms and energy for living organisms.

Carbohydratesf of the diet Starch Sugar Lactose cellulose

Starch Sugar Cellulose

Glucose, the hydrolyzed product of most starch, will be focused in this chapter.

Glucose transport

The fate of absorbed glucose

Section 2 Anaerobic degradation of glucose Glycolysis Pyruvate or lactate Glucose ATP cytosol

2.1 Basic process of glycolysis Glucose Phase 1 Pyruvate Phase 2 Lactate

Phase1 Pyruvate formation from glucose Reaction1 Glucose                                     Glucose-6- Phosphate Hexokinase

Hexokinase O CH2OH OH O CH2OPO3 OH

Hexokinases Hexokinases is a key enzyme in glycolysis and have 4 isoenzymes , isoenzyme 4 present in liver, and named glucokinase. 1 2 3 4 Glucokinase present in liver Hexokinases in all extrahepatic cells

Hexokinase has a low Km 0.1mol/L, high affinity for glucose. Hepatic glucokinase has high Km > 10mol/L, a low affinity for glucose

Phosphohexose isomerase Glucose-6-Phosphate Reaction 2:                                     Fructose-6-Phosphate Glucose-6-Phosphate Phosphohexose isomerase

Phosphohexose isomerase CH2OPO3 O CH2OH OH O CH2OPO3 OH

Reaction 3: Fructose-6-Phosphate                                     Fructose-1,6-Phosphate Phosphofructokinase CH2OPO3 O OH CH2OPO3 O CH2OH OH

Phosphofructokinase Phosphofructokinase

Reaction 4: + helpful Fructose-1,6-Phosphate Glyceraldehyde 3-Phosphate + Dihydroxyacetone Phosphate(DHAP)                                           Aldolase CHO H-C-OH CH2OPO3 CH2OPO3 C=O CH2OH

Aldolase

Reaction 5: + Glyceraldehyde 2 × Glyceraldehyde 3-Phosphate Dihydroxyacetone Phosphate 2 × Glyceraldehyde 3-Phosphate Triose Phosphate Isomerase

Triose Phosphate Isomerase

Reaction 6: Glyceraldehyde 3-Phosphate 1,3-Bisphosphoglycerate C H-C-OH CH2OPO3 ~OPO3 O CHO H-C-OH CH2OPO3 Glyceraldehyde 3-Phosphate Dehydrogenase High energy

Glyceraldehyde 3-Phosphate Dehydrogenase

Reaction 7: 1,3-Bisphosphoglycerate 3-Phosphoglycerate Substrate level phosphorylation 1,3-Bisphosphoglycerate 3-Phosphoglycerate COO H-C-OH CH2OPO3       Phosphoglycerate Kinase C H-C-OH CH2OPO3 ~OPO3 O

Phosphoglycerate Kinase

Reaction 8: 3-Phosphoglycerate 2-Phosphoglycerate Phosphoglycerate Mutase COO H-C-OH CH2OPO3 COO H-C-OPO3 CH2OH

Mutase

Reaction 9: 2-Phosphoglycerate Phosphoenolpyruvate COO COO H-C-OPO3 C~ CH2OH COO C~ CH2 OPO3       Enolase PEP High energy

Enolase

Reaction 10: Phosphoenolpyruvate Pyruvate COO C~ CH2 OPO3 COO C=O CH3       Pyruvate Kinase PEP

Pyruvate Kinase

CO2 + H2O O2 Glucose pyruvate lactate no O2

Conversion of pyruvate to lactate

Conversion of pyruvate to lactate NAD+ NADH + H+ Pyruvate Lactate Lactate dehydrogenase(LDH) COO HO-C-H CH3 COO C=O CH3 NADH + H+ NAD+ L-lactate Pyruvate

How many ATP are produced in above process? 2? 4? Net ATP in glycolysis is 2

The features of the glycolysis pathway Major anaerobic pathway in all cells NAD+ is the major oxidant Requires PO4 Generates 2 ATP’s per glucose oxidized End product is lactate (mammals) Connects with Krebs cycle via pyruvate

2.2 Regulation of Glycolysis

6-phosphofructokinase-1

6-phosphofructokinase-1(PFK-1) Allosteric enzyme negative allosteric effectors Citrate , ATP Positive allosteric effectors AMP, fructose1,6-bisphosphate, fructose2,6-bisphosphate Response to changes in energy state of the cell (ATP and AMP)

Fructose-2,6-bisphosphate Fructose-2,6-bisphosphate , , is a potentially positive effector of PFK-1. formed by phosphorylation of Fructose--6-PO4 catalyzed by PFK-II. Fructose-2,6-bisphosphate Fructose-2,6-bisphosphate

Regulation of Pyruvate Kinase

Allosteric enzyme Regulated by phosphorylation and dephosphorylation Inhibited by ATP. alanine Activated by fructose 1,6 bisphosphate Regulated by phosphorylation and dephosphorylation Inactive Active enzyme PO4

Regulation of Hexokinase Allosteric enzyme Inhibitor: Glucose-6-phosphate except for glucokinase

The Energy Story of Glycolysis Glucose + 2ADP + 2Pi + 2NAD+ 2 Pyruvate + 2ATP + 2NADH + 2H+ + 2H2O Overall ANAEROBIC (no O2) 2Pyruvate + 2NADH Lactate + 2NAD+ Overall AEROBIC(O2) 2NADH 5 ATPs Oxidative phosphorylation

The Significance of Glycolysis Glycolysis is the emergency energy-yielding pathway----ineffient Main way to produce ATP in some tissues red blood cells, retina, testis, skin, medulla of kidney In clinical practice acidosis

Section 3 Aerobic Oxidation of Glucose Oxidation of glucose to pyruvate in cytosol Oxidation of pyruvate to acetylCoA in mitochondria Tricarboxylic acid cycle and oxidative phosphorylation

Oxidation of pyruvate to acetylCoA Pyruvate + CoA       Pyruvate dehydrogenase complex mitochondria This reaction is irreversible.

Pyruvate dehydrogenase complex Comprises of 3 kinds of enzyme and 5 cofactors: E1: pyruvate dehydrogenase E2:dihydrolipoyl transacetylase E3:dihydrolipoyl dehydrogenase Cofactors: Thiamine pyrophosphate(TPP), FAD, NAD, CoA and lipoic acid.

.. .. .. .. .. Pyruvate Dehydrogenase Complex Acetyl-CoA E2 S C-CH3 O HS-CoA S .. H E2 E1 H E3 CH3-C O NAD+ NADH .. acetyl FAD H2 .. TPP CH3-C OH .. hydroxyethyl Pyruvate Dehydrogenase Dihydrolipoyl dehydrogenase Dihydrolipoyl Transacetylase

Tricarboxylic Acid Cycle

Tricarboxylic Acid Cycle Krebs Cycle Tricarboxylic Acid Cycle Citric Acid Cycle All Mean the Same

4 6 4 6 CO2 4 5 CO2 4 4 CARBON BALANCE Oxaloacetate Citrate CH3C O ~ S-CoA CARBON BALANCE 4 Oxaloacetate 6 Citrate 2 carbons in 2 carbons out 4 Malate Isocitrate 6 CO2 TCA cycle 4 Fumarate 5 a-ketoglutarate CO2 4 4 Succinate Succinyl-CoA 8 reactions

Reaction 1. + Coenzyme A Oxaloacetate + Acetyl CoA Citrate Citrate Synthase

Citric Acid or Citrate Citrate Synthase HS-CoA Oxaloacetate (OAA) CH3-C~SCoA O COO- C-OH CH2 -CH2- -OOC COO- C=O CH2 HS-CoA Oxaloacetate CH2COO- HO-C-COO- (OAA) Acetyl-CoA Citric Acid or Citrate

Reaction 2     Isocitrate Citrate Aconitase

Isocitrate Formation Aconitase cis-Aconitate Isocitrate Citrate CH2COO- HO-C-COO- H-C-COO- H CH2COO- C-COO- H CH2COO- H-C-COO- HO-C-COO- H -H2O +H2O cis-Aconitate Citrate Isocitrate Aconitase

-Ketoglutarate + Carbon Dioxide Reaction 3 Isocitrate Isocitrate Dehydrogenase

Isocitrate Dehydrogenase COO- CH2 C=O CH2COO- H-C-COO- HO-C-COO- H CO2 NAD+ NADH + H+ Isocitrate -Ketoglutarate Isocitrate Dehydrogenase

-Ketoglutarate Dehydrogenase Reaction 4 -Ketoglutarate + CoA Succinyl CoA + Carbon Dioxide -Ketoglutarate Dehydrogenase

-Ketoglutarate Succinyl-CoA -Ketoglutarate dehydrogenase Complex NAD+ FAD COO- CH2 C=O -Ketoglutarate Lipoic acid COO- CH2 C~SCoA O HS-CoA TPP CO2 Succinyl-CoA -Ketoglutarate dehydrogenase Complex

ketoglutarate

Reation 5 Succinate + CoA Succinyl CoA Succinyl CoA Synthetase

Succinyl-CoA Synthetase Thioester bond energy conserved as GTP GTP GDP Pi + HS-CoA COO- CH2 C~SCoA O COO- CH2 Succinate Succinyl-CoA Succinyl-CoA Synthetase

Reaction 6 Succinate Fumarate Succinate Dehydrogenase

Reaction 7 Malate Fumarate Fumarase

Reaction 8 Malate Oxaloacetate Malate Dehydrogenase

COOH C COOH C COOH C=O C COOH C FAD FADH2 NAD+ NADH + H+ H2O OH H H Succinate Fumarate Malate Oxaloacetate

4 6 4 6 CO2 4 5 CO2 4 4 CARBON BALANCE Oxaloacetate Citrate CH3C O ~ S-CoA CARBON BALANCE 4 Oxaloacetate 6 Citrate 2 carbons in 2 carbons out 4 Malate Isocitrate 6 CO2 3 NADH 1 FADH2 4 Fumarate 5 a-ketoglutarate CO2 4 4 Succinate Succinyl-CoA GTP

ATP Generated in the Aerobic Oxidation of Glucose There are two ways for producing ATP Substrate level phosphorylation Succinyl CoA to succinate Oxidative phosphorylation

3.2 ATP Generated in the Aerobic Oxidation of Glucose In aerobic oxidation of glucose Gycolysis: 2 NADH and 2ATP produced by substrate level phosphorylation Production of acetylCoA: 2 NADH TCA cycle: 2 ×3NADH ,2× 1 FAD and 2GTP Stoichiometry: 2.5 ATP per NADH 1.5 ATP per FADH Table 6-1 32 ATP are produced for one glucose

Features: Acetyl-CoA enters forming citrate 3 NADH, 1 FADH2, and 1 GTP are formed Oxaloacetate returns to form citrate

3.3 the regulation of aerobic oxidation of glucose The regulation of pyruvate dehydrogenase complex The regulation of tricarboxylic acid cycle

Regulation of Pyruvate Dehydrogenase complex Pyruvate + HS-CoA + NAD+  Acetyl-CoA + NADH + H+ Activators: Inhibitors High NADH means that the cell is experiencing a surplus of oxidative substrates and should not produce more. Carbon flow should be redirected towards synthesis. High Acetyl-CoA means that carbon flow into the Krebs cycle is abundant and should be shut down and rechanneled towards biosynthesis

Mechanism: 1. allosteric regulation 2. Covalent Modification Insulin NADH and acetyl-CoA 2. Covalent Modification E-1 subunits of PDH complex is subject to phosphorylation TPP FAD 1 2 3 Epinephrine Glucagon E1-OH E1-OPO3 H2O HPO4= ATP ADP PDH kinase phosphatase Active Inactive Cyclic-AMP protein kinase Insulin ATP

Regulation of the Citric Acid Cycle Key enzymes : 1. Citrate synthase 2. Isocitrate dehydrogenase 3. α-ketoglutarate dehydrogenase complex Modulators: The ratios of [NADH]/[NAD] and [ATP]/[ADP], high ratios inhibit Additonally, Ca2+ is an activitor Succinyl CoA is a inhibitor summary of TCA

Pentose Phosphate Pathway

PENTOSE PHOSPHATE Pathway Take Home: The PENTOSE PHOSPHATE pathway is basically used for the synthesis of NADPH and D-ribose. It plays only a minor role (compared to GLYCOLYSIS) in degradation for ATP energy.

The primary functions of this pathway are: To generate NADPH, To provide the cell with ribose-5-phosphate.

NADPH differs from NADH physiologically : 1)its primary use is in the synthesis of metabolic intermediates (NADPH as reductant provides the electrons to reduce them), 2) NADH is used to generate ATP

Basic Process Found in cytosol Two phases Oxidative phase nonreversible Nonoxidative phase reversible

The significance of PPP Ribose 5- phosphate: Ribose 5- phosphate is the starting pointing for the synthesis of the nucleotides and nucleic acids.

2) NADPH: a. NADPH is very important ”reducing power”for synthesis of fatty acids and cholesterol, and the synthesis of amino acids via glutamate dehydrogenase. b. In erythrocytes, NADPH is the coenzyme of glutathione reductase to keep the normal level of reduced glutathione Additonally, NADPH serves as the coenzyme of mixed funtion oxidases.

Glycogen Formation and Degradation

Location of glycogen Glycogen is the storage form of glucose in animals and humans Glycogen is synthesized and stored mainly in the liver and the muscles

Features: The structure of glycogen consists of long polymer chains of glucose units connected by an alpha glucosidic bonds. All of the monomer units are alpha-D-glucose, 93% of glucose units are joined by a-1,4-glucosidic bond 7% of glucosyl residues are joined by a-1,6-glucosidic bonds Fig.6-11

Main chains: branch point every 3 units on average. Branch: 5-12 glucosyl residues. Two properties of this structure: 1) High solubility. many terminals 4 hydroxyl groups 2) More reactive points for synthesis and degradation of glycogen.

Glycogen Formation (glycogenesis) Occurs in cytosol of liver and skeletal muscle Dived into 3 phases: ACTIVATION OF D-GLUCOSE GLYCOSYL TRANSFER BRANCHING

Glucose-6- Phosphate Glucose 1. Glucokinase(liver Hexokinase(muscle)                                     Glucokinase(liver Hexokinase(muscle) phosphoglucomutase Glucose-1-phosphate

UDP-Glucose pyrophosphorylase ACTIVATION: UDP-GLUCOSE G-1-P + UTP UDP-GLUCOSE + PPi UDP-Glucose pyrophosphorylase 2 Pi

GLYCOSYL TRANSFER UDPG NON-REDUCING END NEW

BRANCHING Branching Enzyme a1.,4->1,6-glucantransferase Cleave Glycogenin a1.,4->1,6-glucantransferase Branching Enzyme

GLYCOGEN SYNTHESIS ENZYMES UDP-glucose pyrophosphorylase forms UDP-glucose Glycogen Synthase major polymerizing enzyme a1.,4->1,6-glucantransferase

Glycogen Degradation (Glycogenolysis) Glycogenolysis is not the reverse of glycogenesis

Glycogen Synthesis Synthesis Glycogen Degradation Glucose-6-PO4 UDP-Glucose glucose

Phosphorylase and Debranching Enzyme Highly branched core Phosphorylase Phosphorylase Phosphorylase G-1-p Glycogen Debranching enzyme1 Limit Branch ucose Debranching enzyme2 + D-glucose

Hydorlyze a 1,6 branch point Transfer a trisaccharide unit Debranching enzyme: a tandem enzyme glucosidase Oligo α1,4 α 1,4 glucantransferase Hydorlyze a 1,6 branch point Transfer a trisaccharide unit

Glycogen Breakdown Glycogen Phosphorylase and Debranching Enzyme Glucose-1-Phosphate Glucose-6-Phosphate Phosphorylase and Debranching Enzyme PO4 Phosphoglucomutase Glucose Glycolysis Take home: Glycogen contributes glucose to glycolysis and to blood glucose (Liver)

The regulation of glycogensis and glycogenolysis

Regulatory site of glycogenesis and glycogenolysis: Phosphorylase Glycogen synthase

Phosphorylase Phosphorylase G-1-p

Phosphorylase Adenylate cyclase Glucagon,epinephrine Inactive PKA cAMP protein kinase A cAMP b Phosphorylase b kinase Phosphorylase b kinase inactive a Active Phosphorylase

                                                                                      

Glycogen synthase

Glycogen + Glycogen synthase +

Glycogen synthase Adenylate cyclase Glucagon,epinephrine active a cAMP PKA protein kinase A b inactive Glycogen synthase

Phosphorylating inhibitor-1 Adenylyl cyclase Glucagon,epinephrine PKA protein kinase A cAMP synthase phosphorylase b b inactive hydrolyze Phosphorylating inhibitor-1 hydrolyze Active Protein phosphatase-1

Active inactive

Allosteric regulation: Phosphorylase: Activitor: AMP Inhibitor: ATP, glucose-6-phosphate Glycogen synthase: Activitor: ATP, Glucose-6-phosphate

What activates glycogen synthesis inactivates glycogen degradation TAKE HOME: DEGRADATION What activates glycogen degradation inactivates glycogen synthesis. SYNTHESIS What activates glycogen synthesis inactivates glycogen degradation RECIPROCAL REGULATION

The Significance of Glycogenesis and Glycogenolysis Liver maintain blood glucose concentration Skeletal muscle fuel reserve for synthesis of ATP

Glycogen Storage Diseases Deficiency of glucose 6-phosphatase liver phosphorylase liver phosphorylase kinase branching enzyme debranching enzyme muscle phosphorylase Table 6-2

glucogenic amino acids Gluconeogenesis Gluconeogenesis:The process of transformation of non-carbohydrates to glucose or glycogen glucogenic amino acids lactate glycerol organic acids Glucose Glycogen liver, kidney

Ribose 5-PO4 Phosphatase Blood Glucose Glycogen Glucose G6P Kinase F6P F1,6bisP Gly-3-P DHAP 1,3 bisPGA Kinase 3PGA 2PGA PEP Kinase L-lactate Pyruvate OAA

3 irreversible reactions PEP Pyruvate Go’ = -61.9 kJ per mol Go’= -17.2 kJ per mol F-6-PO4 F1,6-bisPO4 Glucose Glucose-6-PO4 Go’= -20.9 kJ per mol Take home: Gluconeogenesis feature enzymes that bypass 3 irreversible KINASE steps

Reaction1 Glucose                                     Glucose-6- Phosphate Hexokinase

Reaction 3 Fructose-6-Phosphate                                     Fructose-1,6-Phosphate Phosphofructokinase

Reaction 10: Phosphoenolpyruvate Pyruvate

3 reactions need to bypass: Pruvate phosphoenolpyruvate Fructose 1,6-bisphosphate fructose 6-phosphate Glucose 6-phosphate glucose

The conversion of pyruvate to phosphoenolpyruvate(PEP) mitochondria CO2 oxaloacetate Pyruvate Pyruvate carboxylase

oxaloacetate malate aspartate cytosol PEP malate oxaloacetate mitochondria aspartate

Mitochondria or cytosol GTP GDP oxaloacetate PEP CO2 Phosphoenolpyruvate carboxykinase

The conversion of Fructose 1,6-bisphosphate to Fructose 6-phosphate Fructose 1,6-bisphosphatase

The conversion of glucose 6-phosphate to Glucose glucose 6-phosphate Glucose Glucose 6-phosphatase

Glucose glucose-6-phosphote Substrate cycle The interconversion of two substrates catalyzed by different enzymes for singly direction reactions is called substrate cycle. Glucose glucose-6-phosphote

Significance: Primarily in the liver (80%); kidney (20%) Maintains blood glucose levels The anabolic arm of the Cori cycle

Cori Cycle

Cori cycle is a pathway in carbohydrate metabolism that links the anaerobic glycolysis in muscle tissue to gluconeogenesis in liver.

Liver is a major anabolic organ L-lactate D-glucose Blood Lactate Blood Glucose THE CORI CYCLE L-lactate D-glucose Muscle is a major catabolic tissue

Significance of cori cycle: avoid the loss of lactate and accumulation of lactate in blood to low blood pH and acidosis. 6 ATP are sonsumed per 2 lactate to glucose

Regulation of gluconeogenesis There are 2 important regulatory points: Fructose 1,6-bisphosphate              Fructose 6-phosphate + Pi Fructose 1,6-bisphosphatase

Fructose 1,6-bisphosphatase Inhibitor: Fructose 2,6-bisphosphate and AMP Activitor: Citrate

To summarize, when the concentration of glucose in the cell is high, the concentration of fructose 2,6-bisphosphate is elevated. This leads to a stimulation of glycolysis . Conversely, when the concentration of glucose is low, the concentration of fructose 2,6-bisphosphate is decreased. This leads to a stimulation of gluconeogenesis. Gluconeogenesis predominates under starvation conditions.

oxaloacetate + ADP + Pi + 2 H+ Pyruvate + CO2 + ATP + H2O              oxaloacetate + ADP + Pi + 2 H+ . pyruvate carboxylase Pyruvate carboxylase is allosterically activated by acetyl CoA

The Significance of Gluconeogenesis Replenishment of glucose and maintaining normal blood sugar level Replenishment of liver glycogen “three carbon” compounds Regulation of Acid-Base Balance Clearing the products lactate, glycerol Glucogenic amino acids to glucose

Blood Sugar and Its Regulation Blood sugar level 3.89-6.11mmol/l Sources of blood sugar---income digestion and absorption of glucose from dietary gluconeogenesis glycogen other saccharides Outcome: aerobic oxidation Glycogen PPP Lipids and amino acids

Regulation of Blood Glucose Concentration Insulin decreasing blood sugar levels Glucagon, epinephrine glucocorticoid increasing blood sugar levels

Insulin The unique hormone responsible for decreasing blood sugar level and promoting glycogen formation, fat, and proteins simultaneously.

The effects of insulin: Effects on membrane actively transport. Effects on glucose utilization Effects on gluconeogenesis.

Glucagon

Epinephrine Stimulates glucogen degradation and gluconeogenesis

Glucocorticoids Inhibit the utilization of glucose Stimulate gluconeogenesis by stimulating protein degradation to liberate amino acids

Review questions

Glucagon

Epinephrine glucocorticoids