Carbohydrate Metabolism and Biological Oxidation Table of Contents

1. Digestion and Absorption of Carbohydrates 2. Hormonal Control of Carbohydrate Metabolism 3.Glycogen Synthesis and Degradation 24.4 Gluconeogenesis 24.5 The Pentose Phosphate Pathway 24.6 Glycolysis 24.7 Terminology for Glucose Metabolic Pathways 24.8 The Citric Acid Cycle 24.9 The Electron Transport Chain 24.10 Oxidative Phosphorylation 11. ATP Production for the Complete Oxidation of Glucose 12. Importance of ATP 13. Non-ETC Oxygen-Consuming Reactions 14. B-Vitamins and Carbohydrate Metabolism

• Carbohydrate digestion: Begins in the mouth – Salivary enzyme “α-amylase” catalyzes the hydrolysis of α- glycosidic linkages of starch and glycogen to produce smaller polysaccharides and disaccharide – maltose – Only a small amount of carbohydrate digestion occurs in the mouth because food is swallowed so quickly into the stomach • In stomach very little carbohydrate is digested: – No carbohydrate digestion enzymes present in stomach – Salivary amylase gets inactivated because of stomach acidity

• The primary site for the carbohydrate digestion is within the small intestine – Pancreatic α-amylase breaks down polysaccharide chains into disaccharide – maltose • The final step in carbohydrate digestion occurs on the outer membranes of intestinal mucosal cells – Maltase – hydrolyses maltose to glucose – Sucrase – hydrolyses sucrose to glucose and fructose – Lactase – hydrolyses lactose to glucose and galactose • Glucose, galactose, and fructose are absorbed into the bloodstream through the intestinal wall. • Galactose and Fructose are converted to products of glucose metabolism in the liver.

• Following absorption the monosaccharides are carried by the portal vein to the liver where galactose and fructose are enzymatically converted to glucose intermediates that enter into the glycolysis pathway • The glucose may then pass into the general circulatory system to be transported to the tissues or converted to glycogen reserve in the liver. • The glucose in the tissues may be

a) oxidized to CO2 and H2O (ATP) b) converted to fat c) converted to muscle glycogen

• Blood-sugar level: – the proper functions of the body are dependent on precise control of the glucose concentration in the blood. – the normal fasting level of glucose in the blood is 70-90 mg/100 ml.

• Abnormal conditions: • A. hypoglycemia – condition resulting from a lower than the normal blood-sugar level (below 70 mg/100 ml) – extreme hypoglycemia, usually due to the presence of excessive amounts of insulin, is characterized by general weakness, trembling, drowsiness, headache, profuse perspiration, rapid heart beat, lowered blood pressure and possible loss ofconsciousness. – Loss of consciousness is most likely due to the lack of glucose in the brain tissue, which is dependent upon this sugar for its energy. • B. hyperglycemia – higher than the normal level (above 120 mg/100 mL); when the pancreas does notsecrete enough insulin – may temporarily exist as a result of eating a meal rich in carbohydrates. – extreme hyperglycemia, the renal threshold (160-170 mg/100 mL) is reached and excess glucose is excreted in the urine

• Besides enzyme inhibition, carbohydrate metabolism may be regulated by hormones • Three major hormones control carbohydrate metabolism: – Insulin ; Glucagon ; Epinephrine • Insulin • 51 amino acid polypeptide secreted by the pancreas • Promotes utilization of glucose by cells • The release of insulin is triggered by high blood-glucose levels • Its function is to lower blood glucose levels by enhancing the formation of glycogen from glucose (glycogen synthesis) • The mechanism for insulin action involves insulin binding to proteins receptors on the outer surfaces of cells which facilitates entry of the glucose into the cells

Copyright © Cengage Learning. All rights reserved 8 Hormonal Control of Carbohydrate Metabolism Glucagon • 29 amino acid peptide hormone produced in the pancreas • Released when blood glucose levels are low • Principal function is to increase blood-glucose concentration by speeding up the conversion of glycogen to glucose (glycogenolysis) in the liver • Glucagon elicits the opposite effects of insulin Epinephrine (also called adrenaline) • Released by the adrenal glands in response to anger, fear, orexcitement • Function is similar to glucagon, i.e., stimulates glycogenolysis • Primary target of epinephrine is muscle cells • Promotes energy generation for quick action

• There are six major metabolic pathways of glucose:

1) Glycogenesis 2) Glycogenolysis 3) Gluconeogenesis 4) Hexose monophosphate shunt 5) Glycolysis 6) Citric Acid Cycle

Glycogenesis and Glycogenolysis • Involved in the regulation of blood glucose concentration • When the dietary intake of glucose exceeds immediate needs, humans and other animals can convert the excess to glycogen, which is stored in either the liver or muscle tissue. • Glycogenesis is the pathway that converts glucose into glycogen. • When there’s need for additional blood glucose, glycogen is hydrolyzed and released into the bloodstream. • Glycogenolysis is the pathway that hydrolyzes glycogen to glucose.

• Metabolic pathway by which glucose is synthesized from non- carbohydrate sources: – The process is not exact opposite of glycolysis • Glycogen stores in muscle and liver tissue are depleted with in 12-18 hours from fasting or in even less time from heavy work or strenuous physical activity • Without gluconeogenesis, the brain, which is dependent on glucose as a fuel would have problems functioning if food intake were restricted for even one day • Gluconeogenesis helps to maintain normal blood-glucose levels in times of inadequate dietary carbohydrate intake • About 90% of gluconeogenesis takes place in the liver • Non-carbohydrate starting materials for gluconeogenesis: – Pyruvate – Lactate (from muscles and from red blood cells) – Glycerol (from triacylglycerol hydrolysis) – Certain amino acids (from dietary protein hydrolysis or from muscle protein during starvation)

Hexose monophosphate shunt

• Initial reactant of the pathway is glucose-6- phosphate • Also termed phosphogluconate pathway, because 6–phosphogluconate is one of the intermediates • A third name is pentose phosphate pathway, because ribose-5-phosphate is one of its products • The main purposes of the HMP shunt are the following: – to produce ribose-5-P for nucleotide synthesis – to produce NADPH from NADP+ for fatty acid and steroid biosynthesis and for maintaining reduced glutathione (GSH) inside erythrocytes – to interconvert pentoses and hexoses

• A series of reactions in the cytoplasm which converts glucose (C6) to two molecules of pyruvate (a C3 carboxylate), and ATP and NADH are produced. • Also called Embden-Meyerhof pathway, after the scientist who elucidated the pathway • an anaerobic process; each

step takes place without O2; one of its advantages, the body can obtain energy from glycolysis quickly, without waiting for a

supply of O2 to be carried to the cells. • occurs in cells lacking mitochondria, e.g., erythrocytes and in certain skeletal muscle cells during intense muscle activity

• Step 1: Formation of glucose-6-phosphate: – Endothermic reaction catalyzed by hexokinase – Energy needed is derived from ATP hydrolysis • Step 2: Formation of Fructose-6-phosphate: – Enzyme: Phosphoglucoisomerase • Step 3: Formation of Fructose 1,6-bisphosphate: – Enzyme: phosphofructokinase • Step 4: Formation of Triose Phosphates: – C6 species is split into two C3 species – Enzyme : Aldolase • Step 5: Isomerization of Triose Phosphates – DHAP is isomerized to glyceraldehyde 3- phosphate – Enzyme: Triosephosphate isomerase • Step 6: Formation of 1,3-bisphosphoglycerate – Glyceraldehyde 3-phosphate is oxidized and phosphorylated – Enzyme: Glyceraldehyde-3-phosphate dehydrogenase

• Step 7: Formation of 3-bisphosphoglycerate – It is an ATP producing step – Enzyme: phosphoglycerokinase • Step 8: Formation of 2-phosphoglycerate – Isomerization of 3-phosphoglycerate to 2-phosphoglycerate – Enzyme: phosphoglyceromutase • Step 9: Formation of Phosphoenolpyruvate: – Enzyme: Enolase • Step 10: Formation of Pyruvate: – High energy phosphate is transferred from phosphoenolpyruvate to ADP molecule to produce ATP and pyruvate – Enzyme: Pyruvate kinase • At this point of carbohydrate metabolism there are at least 2 directions that the product pyruvate may take. • The direction depends primarily upon the availability of oxygen in the cell:

• If there is adequate oxygen, an aerobic pathway is followed and pyruvate enters the Krebs cycle. • If there is insufficient oxygen available, the anaerobic pathway is continued and pyruvate undergoes a series of reactions to produce lactic acid. • Lactic acid then is the end-product of glycolysis, and if there were not some mechanism for its removal, it would accumulate in the muscle cells & cause muscle “crumps”. • Bacteria also use lactate fermentation in the production of yogurt and cheese • Reactions 1  9 are identical for glycolysis and alocoholic fermentation • for pyruvic acid, the crossroads compound, its metabolic fate depends upon the conditions (aerobic or anaerobic) and upon the organism under consideration.

The Cori cycle. Lactate, formed from glucose under anaerobic conditions in muscle cells (glycolysis), is transferred to the liver, where it is reconverted to glucose (gluconeogenesis), which is then transferred back to the muscle cells.

ATP Production and Consumption

• There is a net gain of two ATP molecules in glycolysis for every glucose molecule processed

• Overall equation for glycolysis

+ + Glucose + 2NAD 2 Pyruvate + 2NADH + 2H + 2H2O

2ADP + 2Pi 2ATP

Copyright © Cengage Learning. All rights reserved 22 The Citric Acid Cycle • Citric acid cycle: A series of biochemical reactions in which the acetyl portion of acetyl CoA is oxidized to carbon dioxide and ATP

and the reduced coenzymes FADH2 and NADH are produced • Takes place in the mitochondria • Also known as tricarboxylic acid cycle (TCA) or Krebs cycle: – Named after Hans Krebs who elucidated this pathway • Two important types of reactions: – Reduction of NAD+ and FAD to produce NADH and FADH2 – Decarboxylation of citric acid to produce carbon dioxide – The citric acid cycle also produces 2 ATP by substrate level phosphorylation from GTP

• Step 1: Formation of Citrate • Step 2: Formation of Isocitrate • Step 3: Oxidation of Isocitrate

and Formation of CO2: involves oxidation–reduction as well as decarboxylation • Step 4: Oxidation of Alpha- Ketoglutarate and Formation

of CO2 • Step 5: Thioester bond cleavage in Succinyl CoA and Phosphorylation of GDP to form GTP • Step 6: Oxidation of Succinate • Step 7: Hydration of Fumarate • Step 8: Oxidation of L-Malate to regenerate Oxaloacetate

Copyright © Cengage Learning. All rights reserved 24 The Citric Acid Cycle • Important features of the cycle: • The reactions of the cycle takes place in the mitochondrial matrix, except the succinate dehydrogenase reaction that involves FAD. The enzyme that catalyzes this reaction is an integral part of the inner mitochondrial membrane. • The “fuel “ for the cycle is acetyl CoA, obtained from the breakdown of carbohydrates, fats, and proteins. • Four of the cycle reactions involve oxidation and reduction. The oxidizing agent is either NAD+ (three times) or FAD (once). The operation of the cycle depends on the availability of these oxidizing agents. • In redox reactions, NAD+ is the oxidizing agent when a carbon-oxygen double bond is formed; FAD is the oxidizing agent when a carbon-carbon double bond is formed.

• The three NADH and the one FADH2 that are formed during the cycle carry electrons and H+ to the electron transport chain through which ATP is synthesized. • Two carbon atoms enter the cycle as acetyl unit of the acetyl CoA, and two carbon atoms

leave the cycle as two molecules of CO2. The carbon atoms that enter and leave are not the same ones. The carbon atoms that leave during one turn of the cycle are carbon atoms that entered during the previous turn of the cycle. • Four B vitamins are necessary for the proper functioning of the cycle: riboflavin (in both FAD and α-ketoglutarate dehydrogenase complex), nicotinamide (in NAD+), pantothenic acid (in CoASH), and thiamin (in α-ketoglutarate dehydrogenase complex) • One high-energy GTP molecule is produced by substrate level phosphorylation.

Regulation of the Citric Acid Cycle

• The rate at which the citric acid cycle operates is controlled by ATP and NADH levels • When ATP supply is high, ATP inhibits citrate synthase (Step 1 of Citric acid cycle) • When ATP levels are low, ADP activates citrate synthase • Similarly ADP and NADH control isocitrate dehydrogenase: – NADH acts as an inhibitor – ADP as an activator. 26

• The electron transport chain (ETC) facilitates the passage of

electrons trapped in FADH2 and NADH during citriccycle • ETC is a series of biochemical reactions in which intermediate carriers (protein and non-protein) aid the transfer of electrons and

hydrogen ions from NADH and FADH2 • The ultimate receiver of electrons is molecular oxygen • The electron transport (respiratory chain) gets its name from the fact that electrons are transported to oxygen absorbed via respiration • ETC is the sequence of reactions whereby the reduced forms of the

coenzymes are reoxidized, ultimately by O2

• The enzymes and electron carriers needed for the ETC are located along inner mitochondrial membrane • They are organized into four distinct protein complexes and two mobile carriers • The four protein complexes tightly bound to membrane: • Complex 1: NADH-coenzyme Q reductase • Complex II: Succinate-coenzyme Q reductase • Complex III: Coenzyme Q - cytochrome C reductase • Complex IV: Cytochrome C oxidase • Two mobile electron carriers are: – Coenzyme Q and cytochrome c.

Complex I: NADH- Coenzyme Q Reductase • Facilitates transfer of electrons from NADH to coenzyme Q

Complex II: Succinate- Coenzyme Q Reductase • Succinate is converted to fumarate by this complex • In the process it

generates FADH2 • CoQ is the final recipient of the

electrons from FADH2

Complex III: Coenzyme Q – Cytochrome c Reductase • Several iron-sulfur proteins and cytochromes are electron carriers in this complex • Cytochrome is a heme iron protein in which reversible oxidation of an iron atom occurs Complex IV: Coenzyme Q – Cytochrome c Reductase • The electrons flow from cyt c to

cyt a to cyt a3 • In the final stage of electron transfer, the electrons from cyt + a3, and hydrogen ion (H ) combine with oxygen (O2) to form water + - • O2 + 4H + 4e  2 H2O

• Summary of the flow of electrons through four complexes of the electron transport chain.

• Oxidative phosphorylation – process by which ATP is synthesized from ADP and Pi using the energy released in the electron transport chain by coupled reactions • Coupled Reactions -- are pairs of biochemical reactions that occur concurrently in which energy released by one reaction is used in the other reaction – example: oxidative phosphorylation and the oxidation reactions of the electron transport chain are coupled systems

• The coupling of ATP synthesis with the reactions of the ETC is related to the movement of protons (H+ ions) across the inner mitochondrial membrane • Complexes I, III and IV of ETC chain also serve as “proton pumps” to transfer protons from the matrix side of the inner membrane to the intermembrane space • For every two electrons passed through ETC, four protons cross the inner mitochondrial membrane through complex I, four through complex III and two more though complex IV • This proton flow causes a buildup of H+ in the intermembrane space • The high [H+] in the intermembrane space becomes the basis for ATP synthesis

• The resulting concentration difference (high in intermembrane space than in matrix) constitutes an electrochemical (proton) gradient which is always associated with potential energy • The gradient build-up would spontaneously push the H+ ions through membrane-bound ATP synthase • Proton flow is not through the membrane itself since it is not permeable to H+ ions • The proton flow through the ATP synthetases powers the synthesis of ATP • ATP synthetases are the coupling factors in the ETC/OP coupled reactions

• Formation of ATP accompanies the flow of protons from the intermembrane space back into the mitochondrial matrix. • The proton flow results from an electrochemical gradient across the inner mitochondrial membrane • For each mole of NADH oxidized in the ETC, 2.5 moles of ATP are formed.

• For each mole of FADH2 Oxidized in the ETC, only 1.5 moles of ATP are formed. • For each mole of GTP hydrolyzed one mole of ATP are formed. • Ten molecules of ATP are produced for each acetyl CoA catabolized in the TCA

Summary of the Common Metabolic Pathway

• Cytosolic NADH produced during Step 6 of Glycolysis cannot directly participate in the electron transport chain because mitochondria are impermeable to NADH and NAD+ • Glycerol 3-phosphate- dihydroxyacetone phosphate transport system shuttles electrons from NADH, but not NADH itself, across the membrane: – Dihydroxyacetone phosphate and glycerol phosphate freely cross the mitochondrial membrane – The interconversion shuttles the electrons from NADH to FADH2

• A total of 30 ATP molecules are produced in muscle and nerve cells: – 26 from oxidative phosphorylation / electron transport chain coupled reactions – 2 from oxidation of glucose to pyruvate – 2 from conversion of GTP to ATP • Aerobic oxidation of glucose is 15 times more efficient in the ATP production as compared to anaerobic lactate and ethanol processes • In other cells such as heart and liver cells a more complex shuttle system is used and 32 molecules are produced instead of 30 per glucose molecule

• The cycling of ATP and ADP in metabolic processes is the principal medium for energy exchange in biochemical processes

• >90% of inhaled oxygen via respiration is consumed during oxidative phosphorylation.

• Remaining O2 are converted to several highly reactive oxygen species (ROS) with in the body. • Examples of ROS:

– Hydrogen peroxide (H2O2) - – Superoxide ion (O2 ) and – Hydroxyl radical (OH) – Superoxide ion and hydroxyl radicals have unpaired electron and are extremely reactive • ROS can also be formed due to external influences such as polluted air, cigarette smoke, and radiation exposure

• Reactive oxygen species (ROS) are both beneficial as well a problematic within the body • Beneficial Example: White blood cells produce a significant amount of superoxide free radicals via the following reaction to destroy the invading bacteria and viruses. - + + – 2O2 + NADPH  2O2 + NADP + H

• > 95% of the ROS formed are quickly converted to non toxic species :

Superoxide - + dismutase 2O2 + 2H H2O2 + O2

Catalase 2H2O2 H2O + O2

• About 5% of ROS escape destruction by superoxide dismutase and catalase enzymes.

• Antioxidant molecules present in the body help trap ROS species • Antioxidants present in the body: • Vitamin K • Vitamin C • Glutathione (GSH) • Beta-carotene • Plant products such as flavonoids are also good antioxidants – Have shown promise in the management of many disorders associated with ROS production

• Structurally modified B-vitamins function as coenzymes in the metabolic pathways • Four B Vitamins participate in various reactions: – Niacin – NAD+ and NADH

– Riboflavin – as FAD, FADH2 and FMN – Thiamin – as TPP – Pantothenic acid - as CoA • With out these B-vitamins body would be unable to utilize carbohydrates, proteins and fats as energy sources.