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Chapter 4 Carbohydrates Metabolism The biochemistry and molecular biology department of CMU § 1 Overview • Carbohydrates in general are polyhydroxy aldehydes or ketones or compounds which yield these on hydrolysis. Biosignificance of Carbohydrates • The major source of carbon atoms and energy for living organisms. • Supplying a huge array of metabolic intermediates for biosynthetic reactions. • The structural elements in cell coat or connective tissues. Glucose transporters (GLUT) • GLUT1~5 GLUT1: RBC GLUT4: adipose tissue, muscle The metabolism of glucose • • • • glycolysis aerobic oxidation pentose phosphate pathway glycogen synthesis and catabolism • gluconeogenesis glycogen Glycogenesis Glycogenolysis starch lactate glucose Lactate, amino acids, glycerol H2O+CO2 Pentose phosphate pathway Ribose, NADPH §2 Glycolysis Glycolysis • The anaerobic catabolic pathway by which a molecule of glucose is broken down into two molecules of lactate. glucose →2lactic acid (lack of O2) • All of the enzymes of glycolysis locate in cytosol. 1. The procedure of glycolysis G glycolytic pathway pyruvate lactic acid 1) Glycolytic pathway : G → pyruvate including 10 reactions. (1) G phosphorylated into glucose 6-phosphate HO CH2 H H OH O H OH H H OH ATP ADP 2+ Mg Hexokinase OH G P O CH2 H H OH O H OH H OH H OH G-6-P • Phosphorylated G cannot get out of cell • Hexokinase , HK (4 isoenzymes) , glucokinase, GK in liver ; • Irreversible . Comparison of hexokinase and glucokinase hexokinase glucokinase occurrence in all tissues only in liver Km value 0.1mmol/L 10mmol/L Substrate G, fructose, mannose glucose Regulation G-6-P Insulin (2) G-6-P → fructose 6-phosphate P O CH2 H H OH P O CH2 H H OH OH H O O H OH G-6-P isomerase CH2OH OH OH H OH H F-6-P (3) F-6-P → fructose 1,6-bisphosphate P O CH2 O PFK-1 OH H OH H OH P O CH2 CH2OH H F-6-P H 2+ Mg ATP CH2 O P O OH OH H ADP OH H F-1,6-BP • The second phosphorylation • phosphofructokinase-1, PFK-1 (4) F-1,6-BP → 2 Triose phosphates CH2 O P C O HO C H H C OH CH2 O P C O aldolase H C OH CH2 O P F-1,6-BP • Reversible CH2OH CHO + CHOH CH2 O P dihydroxyacetone glyceraldehyde 3-phosphate, phosphate, GAP DHAP (5) Triose phosphate isomerization CH2 O P C O CH2OH DHAP CHO phosphotriose isomerase CHOH CH2 O P GAP G→2 molecule glyceraldehyde-3-phosphate, consume 2 ATP . (6) Glyceraldehyde 3-phosphate → glycerate 1,3-bisphosphate CHO CHOH CH2 O P glyceraldehyde 3-phosphate Pi NAD+ NADH+H + glyceraldehyde 3-phosphate dehydrogenase, GAPDH O C O~ P CHOH CH2 O P glycerate 1,3-bisphosphate, 1,3-BPG (7) 1,3-BPG → glycerate 3-phosphate O C O~ P CHOH CH2 O P glycerate 1,3-bisphosphate ADP ATP Phosphoglycerate kinase COOCHOH CH2 O P glycerate 3-phosphate • Substrate level phosphorylation (8) Glycerate 3-phosphate → glycerate 2phosphate COO- COO- CHOH CH O P CH2OH CH2 O P glycerate 3-phosphate Mutase glycerate 2-phosphate (9) Glycerate 2-phosphate → phosphoenol pyruvate COOCH O P enolase CH2OH glycerate 2-phosphate COOC O ~ P + H2O CH2 PEP (10) PEP →pyruvate COOC O~ P CH2 PEP ADP ATP pyruvate kinase COOC O CH3 Pyruvate • Second substrate level phosphorylation • irreversible 2) Pyruvate → lactate NADH + H+ COO C O CH3 Pyr NAD+ COO CHOH Lactate dehydrogenase, CH3 LDH Lactic acid Summary of Glycolysis ADP ADP ATP 2+ ATP 2+ Mg Mg F- 6-P F- 1,6-BP G G-6-P HK Isomerase PFK-1 Aldolase lactate NAD+ DHAP GAP LDH H3PO4 NAD+ glyceraldehyde NADH+H+ pyruvate 3-phosphate + NADH+H dehydrogenase ATP pyruvate kinase ADP PEP glycerate 1,3-bisphosphate ADP Enolase ATP Mutase glycerate glycerate H2O 2-phosphate 3-phosphate Phosphoglycerate kinase Total reaction: C6H12O6 + 2ADP + 2Pi 2CH3CHOHCOOH + 2ATP + 2H2O Formation of ATP: The net yield is 2 ~P or 2 molecules of ATP per glucose. 2. Regulation of Glycolysis • Three key enzymes catalyze irreversible reactions : Hexokinase, Phosphofructokinase & Pyruvate Kinase. 1) PFK-1 The reaction catalyzed by PFK-1 is usually the rate-limiting step of the Glycolysis pathway. This enzyme is regulated by covalent modification, allosteric regulation. bifunctional enzyme 2) Pyruvate kinase • Allosteric regulation: F-1,6-BP acts as allosteric activator; ATP and Ala in liver act as allosteric inhibitors; • Covalent modification: phosphorylated by Glucagon through cAMP and PKA and inhibited. ATP Pyruvate Kinase (active) ADP PKA cAMP Glucagon Pyruvate Kinase- P (inactive) 3) Hexokinase and glucokinase • This enzyme is regulated by covalent modification, allosteric regulation and isoenzyme regulation. • Inhibited by its product G-6-P. • Insulin induces synthesis of glucokinase. 3. Significance of glycolysis 1) Glycolysis is the emergency energyyielding pathway. 2) Glycolysis is the main way to produce ATP in some tissues, even though the oxygen supply is sufficient, such as red blood cells, retina, testis, skin, medulla of kidney. • In glycolysis, 1mol G produces 2mol lactic acid and 2mol ATP. § 3 Aerobic Oxidation of Glucose • The process of complete oxidation of glucose to CO2 and water with liberation of energy as the form of ATP is named aerobic oxidation. • The main pathway of G oxidation. 1. Process of aerobic oxidation cytosol first stage G Pyr glycolytic pathway Mitochodria third second stage stage Pyr CH3CO~SCoA CO2 + H2O+ATP TAC 1) Oxidative decarboxylation of Pyruvate to Acetyl CoA COO- NAD+ C O + HSCoA CH3 NADH + H + O Pyruvate dehydrogenase complex pyruvate • irreversible; • in mitochodria. H3C C ~SCoA + CO2 Acetyl CoA Pyruvate dehydrogenase complex: E1 pyruvate dehydrogenase Es E2 dihydrolipoyl transacetylase E3 dihydrolipoyl dehydrogenase thiamine pyrophosphate, TPP (VB1) HSCoA (pantothenic acid) cofactors lipoic Acid NAD+ (Vpp) FAD (VB2) Pyruvate dehydrogenase complex: HSCoA NAD+ The structure of pyruvate dehydrogenase complex N H3C C N NH2 C HC C C H S N C H2 O- + O- C CH2CH2 O P O P C CH3 O O O- TPP H2 C +2H CH (CH2)4 COOH H2C S H2 C S lipoic acid -2H H2C SH CH (CH2)4 COOH SH dihydrolipoic acid HSCoA 4'-phosphopantotheine OH CH3 HS CH2 CH2 NH C CH2 CH2 NH C C O ¦Â-mercaptoethylamine O H ¦Â-alanine OH OH C CH2 O P O P O 3'AMP CH3 O O pantoic acid pyrophosphate pantothenic acid CO2 NADH +H+ NAD+ CoASH 2) Tricarboxylic acid cycle, TCAC • The cycle comprises the combination of a molecule of acetyl-CoA with oxaloacetate, resulting in the formation of a six-carbon tricarboxylic acid, citrate. There follows a series of reactions in the course of which two molecules of CO2 are released and oxaloacetate is regenerated. • Also called citrate cycle or Krebs cycle. (1) Process of reactions CH3CO~SCoA acetyl CoA HSCoA H2O CH2 COO CH2 COO CO COO CH2 COO oxaloacetate NADH+H+ H2O HO C citrate synthase COO CH2 COO malate dehydrogenase CH COO aconitase NAD HO CH COO malate CH2 COO fumarase HC COO Citrate cycle isocitrate CH HO CH CH2 COO COO COO + NAD fumarate isocitrate dehydrogenase succinyl CoA syntetase CH2 COO NADH+H+ NAD+ succinate ADP CO~ SCoA CH2 COO CH2 CH2 CoASH GTP GDP+Pi CO2 NADH+H+ succinate dehydrogenase FAD CH2 COO H2O CH2 COO OOC CH FADH2 COO cis-aconitate citrate + H2O C aconitase CO2 HSCoA COCOO succinyl-CoA ¦Á-ketoglutarate ¦Á-ketodehydrogenase glutarate ATP complex Summary of Krebs Cycle ① Reducing equivalents ② The net reaction of the TCAC: acetylCoA+3NAD++FAD+GDP+Pi+2H2O → 2CO2+3NADH+3H++FADH2+GTP+ HSCoA ③ Irreversible and aerobic reaction ④ The enzymes are located in the mitochondrial matrix. ⑤ Anaplerotic reaction of oxaloacetate ADP + Pi ATP CH3 Biotin C H2 C O + CO2 pyruvate carboxylase COOH CH3 COOH C O COOH NADPH+H+ C O + CO2 COOH NADP+ COOH NAD+ NADH+H+ C H2 C H2 malic enzyme CHOH COOH COOH malic acid DH C O COOH (2) Bio-significance of TCAC ① Acts as the final common pathway for the oxidation of carbohydrates, lipids, and proteins. ② Serves as the crossroad for the interconversion among carbohydrates, lipids, and non-essential amino acids, and as a source of biosynthetic intermediates. Krebs Cycle is at the hinge of metabolism. 2. ATP produced in the aerobic oxidation • acetyl CoA → TCAC : 3 (NADH+H+) + FADH2 + 1GTP → 12 ATP. • pyruvate →acetyl CoA: NADH+H+ → 3 ATP • 1 G → 2 pyruvate : 2(NADH+H+) → 6 or 8ATP 1mol G: 36 or 38mol ATP (12+3 )×2 + 6( 8 )=36( 38 ) 3. The regulation of aerobic oxidation • The Key Enzymes of aerobic oxidation The Key Enzymes of glycolysis Pyruvate Dehydrogenase Complex Citrate synthase Isocitrate dehydrogenase (rate-limiting ) -Ketoglutarate dehydrogenase (1) Pyruvate dehydrogenase complex allosteric activators: AMP, CoA, NAD+,Ca2+ allosteric inhibitors: ATP, acetyl CoA, NADH, FA Pyruvate dehydrogenase (active form) Pi ATP pyruvate dehydrogenase phosphatase H2O Ca2+,insulin pyruvate dehydrogenase kinase ADP pyruvate dehydrogenase P (inactive form) acetyl CoA, NADH ADP, NAD+ (2) Citrate synthase • Allosteric activator: ADP • Allosteric inhibitor: NADH, succinyl CoA, citrate, ATP (3) Isocitrate dehydrogenase • Allosteric activator: ADP, Ca2+ • Allosteric inhibitor: ATP (4) -Ketoglutarate dehydrogenase • Similar with Pyruvate dehydrogenase complex Oxidative phosphorylation→TCAC↑ • ATP/ADP↑ inhibit TCAC, Oxidative phosphorylation ↓ • ATP/ADP↓,promote TCAC, Oxidative phosphorylation ↑ 4. Pasteur Effect • Under aerobic conditions, glycolysis is inhibited and this inhibitory effect of oxygen on glycolysis is known as Pasteur effect. • The key point is NADH : NADH mitochondria Pyr TCAC CO2+H2O Pyr can’t produce to lactate. §4 Pentose Phosphate Pathway 1. The procedure of pentose phosphate pathway/shunt In cytosol 1) Oxidative Phase NADP + NADPH+H+ H2O 6-phosphogluco6-Phosphogluconate G-6-P G-6-P nolactone 6-Phospho dehydrogenase NADP+ gluconolactonase 6-phosphogluconate dehydrogenase Ribose 5-P Isomerase Xylulose 5-P NADPH+H+ CO2 Ribulose 5-P Epimerase 2) Non-Oxidative Phase Ribose 5-p Fructose 6-p Glycolysis Fructose 6-p Xylulose 5-p Xylulose 5-p Glyceraldehyde 3-p • Transketolase: requires TPP • Transaldolase The net reation: 3G-6-P + 6NADP+ → 2F-6-P + GAP + 6NADPH + H+ + 3CO2 2. Regulation of pentose phosphate pathway Glucose-6-phosphate Dehydrogenase is the rate-limiting enzyme. NADPH/NADP+↑, inhibit; NADPH/NADP+↓, activate. 3. Significance of pentose Phosphate pathway 1) To supply ribose 5-phosphate for biosynthesis of nucleic acid; 2) To supply NADPH as H-donor in metabolism; NADPH is very important “reducing power” for the synthesis of fatty acids and cholesterol, and amino acids, etc. NADPH is the coenzyme of glutathione reductase to keep the normal level of reduced glutathione; H2O2 2GSH NADP+ glutathione reductase 2H2O G-S-S-G NADPH + H+ So, NADPH, glutathione and glutathione reductase together will preserved the integrity of RBC membrane. Deficiency of glucose 6-phosphate dehydrogenase results in hemolytic anemia. favism NADPH serves as the coenzyme of mixed function oxidases (monooxygenases). In liver this enzyme participates in biotransformation. §5 Glycogen synthesis and catabolism Glycogen is a polymer of glucose residues linked by (1→4) glycosidic bonds, mainly (1→6) glycosidic bonds, at branch points. 1. Glycogen synthesis (Glycogenesis) • The process of glycogenesis occurs in cytosol of liver and skeletal muscle mainly. ATP ADP G G-6-P HK or GK UDP Gn+1 G-1-P UDPG glycogen UDPG synthase pyrophosphorylase UTP PPi Gn • UDPG: G active pattern, G active donor. • In glycogen anabolism, 1 G consumes 2~P. • Glycogen synthase: key E. O CH2OH HN O H H OH H O H O OH H OH P O O O O P O CH2 O O H H OH H OH H UDPG N Branching enzyme 2. Glycogen catabolism (glycogenolysis) Pi Gn-1 Gn H2O G-1-P Phosphorylase G-6-P Pi G-6-Pase G Phosphorylase: key E; The end products: 85% of G-1-P and 15% of free G; There is no the activity of glucose 6phosphatase (G-6-Pase) in skeletal muscle. Debranching enzyme: glucan transferase -1,6-glucosidase (1→6) linkage Nonreducing ends Glycogen phosphorylase Transferase activity of debranching enzyme (1→6) glucosidase activity of debranching enzyme Glucose 3. Regulation of glycogenesis and glycogenolysis 1) Allosteric regulation In liver: G phosphorylase glycogenolysis In muscle: AMP Ca2+ phosphorylase-b ATP G-6-P phosphorylase-a glycogenolysis 2) Covalent modification Glucagon epinephrine cAMP receptor PKA G protein Adenylyl cyclase Phosphorylase Glycogen synthase glycogenolysis glycogenesis Blood sugar glucagon, epinephrine active adenylate cyclase inactive adenylate cyclase cAMP phosphorylase b kinase ATP ATP inactive PKA ATP active PKA glycogen synthase (active) glycogen synthase (inactive) Pi H2O ATP H2O phosphorylase b kinase ATP P inhibitor-1 (inactive) P ADP ADP Pi ADP P phosphorylase b phosphorylase a Pi protein phosphatase-1 inhibitor-1 (active) P H2O §6 Gluconeogenesis • Concept: The process of transformation of noncarbohydrates to glucose or glycogen is termed as gluconeogenesis. • Materials: lactate, glycerol, pyruvate and glucogenic amino acid. • Site: mainly liver, kidney. 1. Gluconeogenic pathway • The main pathway for gluconeogenesis is essentially a reversal of glycolysis, but there are three energy barriers obstructing a simple reversal of glycolysis. 1) The shunt of carboxylation of Pyr GDP CO 2 COO - CH O ~ P PEP carboxykinase GTP COO £¨ 1/3Mt. . 2/3cytosal£© - CH2 PEP ADP Pyr kinase ATP ADP+Pi ATP CO COO 2 C O Biotin C O CH2 Pyr carboxylase CH3 COOH £¨ Mt.£© pyruvate oxaloacetic acid 2) F-1, 6-BP →F-6-P ATP ADP PFK-1 F-6-P Fructosebisphosphatase Pi H2O F-1,6-BP 3) G-6-P →G ATP ADP HK G Glucose-6phosphatase H2 O Pi • 2 lactic acid G-6-P G consume ATP? glucose glycogen G-6-P G-1-P gluconeogenesis CYTOSOL MITOCHONDRIA F-6-P NAD+ glyceraldehyde 3-P glutamate glutamate ¦Á-ketoglutarate NADH+H+ DHAP malic acid malic acid F-1,6BP OAA Asp ¦Á-ketoglutarate NADH+H+ Asp 1.3-bisphospho2/3 glycerate CO2 ADP GDP ATP glycerate 3-P phosphoenol pyruvate ADP glycerate 2-P PK ATP NAD+ NADH+H+ lactate pyruvate OAA GTP GTP glycerol NAD+ ADP CO2 GDP 1/3 phosphoenol pyruvate ATP CO2 pyruvate 2. Regulation of gluconeogenesis • Substrate cycle: The interconversion of two substrates catalyzed by different enzymes for singly direction reactions is called “substrate cycle”. • The substrate cycle produces net hydrolysis of ATP or GTP.------futile cycle Key enzymes of gluconeogenesis PEP carboxykinase Pyr carboxylase Fructose-bisphosphatase Glucose-6-phosphatase gluconeogenesis: F-6-P Pi F-2,6-BP PFK-1 FBPase-1 AMP H2O ATP F-1,6-BP glycolysis ADP F-2,6-BP glucagon PEP ADP F-1,6-BP glucagon Ala in liver insulin OAA ATP Pyr acetyl CoA 3. Significance of gluconeogenesis (1) Replenishment of Glucose by Gluconeogenesis and Maintaining Normal Blood Sugar Level. (2) Replenishment of Liver Glycogen. (3) Regulation of Acid-base Balance. First stages (cytosol) Second stages (Mt.) Third stages (Mt.) Lactic acid (Cori) cycle • Lactate, formed by the oxidation of glucose in skeletal muscle and by blood, is transported to the liver where it re-forms glucose, which again becomes available via the circulation for oxidation in the tissues. This process is known as the lactic acid cycle or Cori cycle. • prevent acidosis;reused lactate Lactic acid cycle glucose glucose gluconeogenesis glucose glycolytic pathway pyruvate pyruvate NADH+H+ NAD+ NAD+ NADH+H+ lactate liver lactate lactate blood muscle §6 Blood Sugar and Its Regulation 1. The source and fate of blood sugar origin (income) fate (outcome) CO2 + H2O + energy dietary supply liver glycogen non-carbohydrate (gluconeogenesis) other saccharides blood sugar glycogen 3.89¡« 6.11mmol/L other saccharides non-carbohydrates (lipids and some amino acids) >8.89¡« 10.00mmol/L (threshold of kidney) urine glucose Blood sugar level must be maintained within a limited range to ensure the supply of glucose to brain. The blood glucose concentration is 3.89~ 6.11mmol/L normally. 2. Regulation of blood sugar level 1)insulin: for decreasing blood sugar levels. 2)glucagon:for increasing blood sugar levels. 3)glucocorticoid: for increasing blood sugar levels. 4)adrenaline:for increasing blood sugar levels. 3. Abnormal Blood Sugar Level • Hyperglycemia: > 7.22~7.78 mmol/L • The renal threshold for glucose: 8.89 ~10.00mmol/L • Hypoglycemia: < 3.33~3.89mmol/L Pyruvate as a junction point