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Chapter 16 Glycolysis and Gluconeogenesis Outline 16.1. Glycolysis Is an Energy-Conversion Pathway in Many Organisms 16.2. The Glycolytic Pathway Is Tightly Controlled 16.3. Glucose Can Be Synthesized from Noncarbohydrate Precursors 16.4. Gluconeogenesis and Glycolysis Are Reciprocally Regulated 2 Trioses (three carbons) •Monosaccharides, the simplest carbohydrates, are aldehydes (醛類) or ketones (酮類) that have two or more hydroxyl groups (氫氧基;羥基) •The smallest monosaccharides, composed of three carbon atoms, are dihydroxyacetone and D- and L- glyceraldehyde Contains a keto group Contains a aldehyde group •Simple monosaccharides with four, five, six, and seven carbon atoms are called tetroses, pentoses, hexoses, and heptoses (suffix –ose means sugar) Sugar Stereochemistry Horizontal = forward Most sugars are in the D form Vertical = back Fisher projections: OH group is in the right Berg et al. Biochemistry D isomer L isomer Sugar Stereochemistry Asymmetric C farthest from the aldehyde (or ketone) group D-glucose Hemiacetal formation •Open-chain forms of these sugars cyclize into rings. In general, an aldehyde can react with an alcohol to form a hemiacetal Hemiketal formation •A ketone can react with an alcohol to form a hemiketal Haworth Structure of Glucose •For an aldohexose such as glucose, a single molecule provides both the aldehyde and the alcohol : the C-1 aldehyde in the open-chain form of glucose reacts with the C-5 hydroxyl group to form an intramolecular hemiacetal •The resulting cyclic hemiacetal, a six-membered ring, is called pyranose because of its similarity to pyran Haworth Structure of Fructose •The C-2 keto group in the open-chain form of a ketohexose, such as fructose, can form an intramolecular hemiketal by reacting with either the C-6 hydroxyl group to form a six-membered cyclic hemiketal or the C-5 hydroxyl group to form a five-membered cyclic hemiketal •The five-membered ring is called a furanose because of its similarity to furan Some fates of glucose •Glycolysis 糖解作用: metabolizes one molecule of glucose to two molecules of pyruvate with the concomitant net production of two molecules of ATP. – The process is anaerobic, O2 is not required – Pyruvate is further processed: •Anarobically through fermentation 發酵作用, two molecules of ATP. •Aerobically by complete oxidation to CO2, generating more ATP Alcoholic fermentation : glucose to ethanol 2ATP 6C 2ATP More ATP 2 x 3C Lactic acid fermentation : glucose to lactate 10 Glycolysis • Glycolysis: • Convert one glucose to two pyruvate, producing two ATP and two NADH • Derived from the Greek stem glyk, “sweet”; and the word lysis, “dissolution” • also known as the EmbdenMeyerhof pathway • Common to virtually all cells • In eukaryotes, occurs in cytoplasm • 2 stages, 10 steps 11 Gluconeogenesis 糖質新生 • Glucose can be synthesized from noncarbohydrate precursors, such as pyruvate and lactate, in the process of gluconeogenesis • Glycolysis and gluconeogenesis have some of the same enzymes in common, the two pathways are not simply the reverse of each other. The highly exergonic, irreversible steps of glycolysis are bypassed in gluconeogenesis. 12 Glucose is generated from dieatry carbohydrates Starch Complex carbohydrate Glycogen (肝醣) Pancreatic α-amylase (澱粉酶) Salivary α-amylase (Amylase cleaves the α -1,4 bonds, but not α -1,6 bond) Maltose Maltotriose simpler carbohydrate Maltase Maltose two glucoses (麥芽三糖) α-glucosidae Digest maltotriose and others Limit dextrin (極限糊精; 指上述無法再 被分解之α-1,6 bond 的糖) α -Dextrinase Digest limit dextrin Sucrase Sucrose fructose + glucose lactase Lactose glucose + galactose monosaccharides Absorption by the intestine Transport in the blood 13 16.1. Glycolysis Is an Energy-Conversion Pathway in Many Organisms •Glycolysis is common to all cells, both prokaryotic and eukaryotic. – Take place in the cytoplasm – Divided into two stage •The trapping and preparation phase (Energy investment phase) – Conversion of glucose into fructose 1,6-biphosphate – Consists of three step » Phosphorylation » Isomerization » Second phosphorylation – The cleavage of fructose 1,6-bisphosphate into two glyceraldehyde 3-phosphate •ATP is generated (energy payoff phase) – glyceraldehyde 3-phosphate are oxided to pyruvate 14 First stage of glycolysis 15 Mg2+ Mg2+ 2 molecules Fig 16.2 Stages of glycolysis 16 glyceraldehyde 3-phosphate Mg2+ Mg2+ Mg2+ K+ Mg2+ K+ Fig 16.2 Stages of glycolysis 17 Hexokinase traps glucose in the cell and begins glycolysis • Glucose enters cells through specific transport proteins • It is phosphorylated by ATP to form glucose 6phosphate – G-6P cannot pass through the membrane – Phosphoryl group destabilized glucose, thus facilitating metabolism Mg2+ 一般而言, kinase的 反應大都需要Mg2+ 此反應大致屬不可逆反應 18 8Å • Glucose induced hexokinase structural changes • Substrate-induced cleft closing is general feature of kinase Glucose 與hexokinase結合後會使酵素更靠近(induced fit), glucose周圍環境更極性,促使glucose與ATP反應,同時也 避免與水分子接觸,而阻擾ATP的反應。 Fig 16.3 Induced fit in hexokinase 19 Fructose 1,6-bisphosphate is generated from glucose 6-phosphate • The isomerization of glucose 6-phophate to fructose 6-phosphate – Conversion of an aldose into ketose – Catalyzed by phosphoglucose isomerase – Reversible reaction 1 6 2 1 1 Enzyme binding (His) and open the ring Aldehyde group Dissociation and closing of the ring 2 Keto group 20 • Fructose 6-phosphate is phosphorylated at the expense of ATP to fructose 1,6-bisphosphate – Catalyzed by phosphofructokinase (PFK) • Play a central role in the metabolism • 當ATP缺乏或ADP、AMP增多時,此酵素活性會被活化 Mg2+ 只要形成F-1,6-BP, 意味反應只進入glycolysis •bis- means two separate monophosphoryl groups •di- means two phosphoryl groups are present and are connected by an anhydride bond 21 The six-carbon is cleaved into two three-carbon fragments • Fructose 1,6 bisphosphate is cleaved into glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) – Catalyzed by aldolase (醛縮酶) 1 2 3 4 5 6 Ketos aldose • At equilibrium, 96% DHAP •GAP for the subsequent reactions of glycolysis 22 Fig 12-5 The class I aldolase reaction. 23 Principles of Biochemistry Lehninger Mechanism: trisoe phosphate isomerase slavages a three-carbon fragment Fig 16.4 structure of triose phosphate isomerase Fig 16.5 Catalytic mechanism of triose phosphate isomerase 作用機制與glycolysis中 phosphoglucose isomerase相似 **Triose phosphate isomerase (TPI) has two noteworthy feature • Kinetically perfect enzyme: 作用很快 24 • Suppresses an undesired side reaction: the active site is kept closed until the desirable reaction takes place The oxidation of an aldehyde to an acids powers the formation of a compound with high phosphoryl-transfer potential 1 molecule of glucose (6C) 2 molecule of GAP (3C) • The conversion of glyceraldehyde 3phosphate into 1,3-bisphosphoglycerate (1,3-BPG) – Catalyzed by glyceraldehyde 3phosphate dehydrogenase Energy payoff stage High phosphoryl-transfer potential 25 Second stage of glycolysis • Glyceraldehyde 3 –phosphate dehydrogenase – Oxidation of the aldehyde to a carboxylic acid by NAD+ – Joining of the carboxylic acid and orthophosphate to form the acyl-phosphate product. 26 Linked to the enzyme by a thioester bond 不利反應進行 Fig 16.6 Free-energy profiles for glyceraldehyde oxidation followed by acylphosphate formation. 27 His 176 Cys 149 Positive charge polarized the thioester intermediate to facilitate the attack by orthophosphate Fig 16.8 Catalytic mechanism of glyceraldehyde28 3phosphate dehydrogenase ATP is formed by phosphoryl transfer from 1,3bisphosphaoglycerate Mg2+ Substrate-level phosphorylation is a type of chemical reaction that results in the formation and creation of adenosine triphosphate (ATP) by the direct transfer and donation of a phasphate group to adenosine triphosphate (ADP) from a reactive intermediate. 29 rearrangement dehydration Enol phosphate has a high phosphoryl-transfer potential 30 • Mutase: catalyzed the intramolecular shift of a chemical group, such as a phosphoryl group Enz-His-phosphate + 3-phosphoglycerate Enz-His + 2,3-bisphosphoglycerate • Mutase functions as a phosphatase Enz-His + 2,3-bisphosphoglycerate Enz-His-phosphate + 2-phosphoglycerate Mg2+ 31 Pyruvate kinase Tautomerizes rapidly and nonenzymatically K+, Mg2+ or Mn2+ More stable ketone 32 Two ATP molecules are formed in the conversion of glucose into pyruvate The net reaction of gylcolysis: Glucose + 2Pi + 2 ADP +2NAD+ 2 pyruvate + 2ATP + 2 NDAH +2H+ + 2H2O Energy release -96kJ/mol 33 NAD+ is regenerated from the metabolism of pyruvate Fermentation (absence of O2) 34 1. Alcohol fermentation Ethanol is formed from pyruvate in yeast and several other microorganisms Require coenzyme thiamine pyrophosphate (TPP) and Mg2+ Glucose + 2Pi + 2 ADP +2H+ 2 ethanol + 2CO2 + 2ATP + 2H2O (No NAD+ and NADH in this equation) 35 2. Lactic acid fermentation Lactate is formed from pyruvate in microoragnisms , higher organism (O2 limiting), as in muscle cells (intense activity) Glucose + 2Pi + 2 ADP 2 lactate + 2ATP + 2H2O 36 37 The binding site for NAD+ is similar in many dehydrogenase Glyceraldehyde 3-phosphate dehydrogenase Alcohol dehydrogenase Lactate dehydrogenase •Different 3-D structure •NAD+-binding site similar Rossmann fold Fig 16.12 NAD+-binding region in dehydrogenases. 38 Fructose and galactose are converted into glycolytic intermediates •There are no catabolic pathway for metabolizing fructose or galactose convert these sugars into a metabolite of glucose Fig 16.13 Entry points in glycolysis for galactose and fructose 39 1. Fructose 1-phosphate pathway in liver 2. Fructose can be phosphorylated to fructose 6-phosphate by hexokinase glycolysis Fig 16.14 Fructose metabolism 40 Galactose is converted into glucose 6-phosphate in 4 steps: • Galactose-glucose interconversion pathway 41 phosphoglucomutase Glucose 6-phosphate Glycolysis Galactose + ATP 42 glucose 1-phosphate + ADP + H+ Many adults are intolerant of milk because they are different in lactase • Lactose intolerance or hypolactasia 乳糖不耐症 – A deficiency of the enzyme lactase • Lactose is a good energy for microorganisms in colon and produce CH4 and H2 • Uncomfortable feeling of gut distension and flatulence • diarrhea 在缺乏乳糖酶的情況下,人攝入的乳糖不能被消化吸收進血液,而是滯留在腸道。 腸道細菌發酵分解乳糖的過程中會產生大量氣體而造成腹脹、放屁。過量的乳糖還 會升高腸道內部的滲透壓,阻止對水分的吸收而導致腹瀉。 當未分解吸收的乳糖進 入結腸後,被腸道存在的細菌發酵成為小分子的有機酸如醋酸、丙酸、丁酸等,並 產生一些氣體如甲烷、H2、CO2等,這些產物大部分可被結腸重吸收。新生兒小腸粘 43 膜乳糖酶缺乏是主要病因,部分人群因長期不攝入奶及奶製品也會造成。 Galactosemia 半乳糖血症 • Inherited deficiency in galactose 1-phosphate uridyl transferase activity – 血中半乳糖濃度增加,並可於尿液中測出 – 出生時並無異狀,餵乳數天後發生嚴重吐奶、呈昏睡 狀,之後會有肝脾腫大、黃疸,嚴重的患者會因血液 感染而死亡。 – 治療方式乃移除食物中的半乳醣(乳糖) – 易造成白內障 • 形成galactitol (醣醇)的堆積 44 16.2 The Glycolytic Pathway Is Tightly Controlled • In metabolic pathways, enzymes catalyzing essentially irreversible reactions are potential site of control – Phsophofructokinase, Hexokinase and Pryuvate kinase • The control of glycolysis in two tissues – skeletal muscle and liver – The primary control of muscle glycolysis is the energy charge of the cell – The ratio of ATP to AMP – The regulation of glycolysis in the liver illustrates the biochemical versatility of the liver 45 The control of Muscle glycolysis • Phsophofructokinase – The most important control site in the mammalian glycolytic pathway – High levels of ATP allosterically inhibit the enzyme • ATP 會binding 至regulatory site, 而降低酵素與fructose6-phosphate的affinity • ATP/AMP ratio lower enzyme activity increase – Decrease in pH inhibit enzyme activity • 肌肉在缺氧狀態下會產生過多乳酸,致使pH值下降,因 此抑制糖解作用,以避免肌肉累積過多的酸。 Fig 16.16 structure of phosphofructokinase Fig 16.17 allosteric regulation 46 of phosphofructokinase Alanine Fig 16.18 Regulation of glycolysis in muscle 47 The regulation of glycolysis in the liver •Liver has more-diverse biochemical functions than muscle –Maintain blood-glucose levels •Stores glucose as glycogen, and releases glucose –Use glucose to generate reducing power for biosynthesis •Phosphofructokinase –ATP regulation –Low pH is not a metabolic signal for liver enzyme –Inhibited by citrate (TCA cycle intermediate) •High level citrate in the cytoplasm biosynthesis precusor •Enhancing the inhibitory effect of ATP –Fructose 2,6-bisphosphate as a potent activator 48 血糖濃度增加 fructose-6-phosphate 增加 加速fructose-2,6-phosphate合成 增加phosphofructokinase affinity Feedforward stimulation Fructose 2,6-bisphosphate increase the affinity of phosphofructokinase and diminishes the inhibitory of ATP Fig 16.19 Regulation of phosphofructokinase by fructose 2,6-bisphosphate Fig 16.20 Activation of phosphofructokinase by fructose 2,6-bisphosphate •[ATP] increase allosteric inhibitor •The inhibitory effect of ATP is reversed by F-2,6-BP 49 Hexokinase • The hexokinase reaction in the liver is controlled as in the muscle • Another isozyme of hexokinase -- Glucokinase, is not inhibited by glucose 6-phsophate – Phosphorylates glucose only when the glucose is abundant (the affinity for glucose is about 50-fold lower than hexokinase) – Provide glucose 6-phosphate for the synthesis of glycogen and for the formation of fatty acid • In pancreas β cells, when blood-glucose levels are elevated – glucokinase increased formation of glucose 6-phosphate – Leads to the secretion of the insulin • Remove glucose from the bloods for storage at glycogen or 50 conversion into fat Pyruvate kinase (a tetramer of 57kd subunits) • L type pryuvate kinase predominates in the liver, M type in muscle and the brain – have many properties in common L type pyruvete kinase The glucagon昇糖素triggered cyclic AMP cascade Phosphorylation of pyruvate kinase Diminish activity 由賀爾蒙誘使的磷酸化, 使肝臟不再消耗可提供腦及 肌肉之葡萄糖 Fig 16.21 control of the catalytic activity of pyruvate kinase 51 昇糖素 cAMP-dependent protein kinase Protein phosphatase Regulation of pyruvate kinase 52 A family of transporters enables glucose to enter and leave animal cells • Glucose transporters – GLUT1 to GLUT5 – 500 residues of a single polypeptide chain – 12-transmembrane-helix structure Normal serum-glucose level: 4mM to 8mM 當血糖過多時,才會將glucose送入 53 Mg2+ Mg2+ 2 molecules Fig 16.2 Stages of glycolysis 54 glyceraldehyde 3-phosphate Mg2+ Mg2+ Mg2+ K+ Mg2+ K+ Fig 16.2 Stages of glycolysis 55 16.3 Glucose can be synthesized from noncarbohydrate precusor • Gluconeogenesis : the synthesis of glucose from noncarbohydrate precusor – Especially important during a longer period of fasting or starvation – Noncarbonhydrate precusor converted into pyruvate and then synthesis of glucose – Major site is the liver (small amount in the kidney, little in the brain, skeletal muscle or heart muscle) 56 57 Fig 16.24 Pathway of gluconeogenesis 58 Fig 16.24 Pathway of gluconeogenesis • The major noncarbohydrate precusors for gluconeogenesis are: –Lactate •Lactate is formed by active skeletal muscle •Readily converted into pyruvate by the action of lactate dehydrogenase –amino acids •Derived from proteins in the diet and during starvation, from the breakdown of proteins in skeletal muscle –Glycerol •The hydrolysis of triacylglycerols in fat cells 59 Gluconeogenesis is not a reversal of glycolysis Glycolysis Glucose +ATP hexokinase Glucose 6-phosphate + H2O glucose 6phosphatase glucose + Pi glucose-6-phsophate + ADP ΔG=-33kJ/mol Froctose 1,6bisphosphatase Fructose-6-phosphate +ATP phosphofructokinase flucose-1,6-bisphsophate + ADP ΔG=-22kJ/mol phosphoenolpyruvate +ADP Pyruvate kinase pyruvate + ATP ΔG=-17kJ/mol Fructose 1,6-bisphosphate + H2O Fructose 1,6-bisphosphate + Pi Phosphoenolpyruvate carboxykinase Oxaloacetate + GTP Phosphoenolpyruvate + GDP+ CO2 pyruvate + CO2+ ATP + H2O Pyruvate carboxylase Oxaloacetate +ADP+Pi+2H+ Gluconeogenesis 60 The conversion of pyruvate into phosphoenolpyruvate begins with the formation of oxaloacetate Carboxylation Pyruvate carboxylase Inside the mitochondria Decarboxylation & phosphorylation phosphoenolpyruvate carboxylase Cytoplasm Pyruvate + ATP + GTP +H2O phosphoenolpyruvate + ADP +GDP+ Pi +2H+ 61 Pyruvate carboxylase ATP activation domain Biotin covalently attached, serves as a carrier of activated CO2 Fig 16.25 Domain structure of pyruvate carboxylase ε-amino group (amide bond) N1 atom Carboxybiotin-enzyme intermediate Long, flexible chain Biotin is not carboxylated unless acetyl Co-A is bound 一般而言, CO2在身體中通常以HCO3-形式存在 • The carboxylation of pyruvate take place in three stage – HCO3- + ATP HOCO2-PO32- + ADP – Biotin-Enzyme + HOCO2-PO32- CO2-biotin-enzyme + Pi – CO2-biotin-enzyme + pyruvate biotin-enzyme + oxaloacetate 62 Oxaloacetate is shuttled into the cytoplasm and converted into phosphoenolpyruvate •Oxaloacetate must be transported to the cytoplasm to complete the pathway –Oxaloacetate is reduced to malate •by NADH-linked malate dehydrogenase in the mitochondria –Malate transported across the mitochondrial –Reoxidized to oxaloacetate •by NAD+-linked malate dehydrogenase in the cytoplasm phosphoenolpyruvate carboxylase 63 The generation of free glucose is an important control point •In most tissues, gluconeogenesis ends in glucose 6-phosphate – Could form glycogen – Not transported out of the cell •The final step in the generation of glucose does not in the cytoplasm – Glucose 6-phosphate is transported into the lumen of the endoplasmic reticulum – Hydrolyzed to glucose by glucose 6-phosphatase •Bound to the membrane •Ca2+-binding stabilizing protein is essential for enzyme activity – Glucose and Pi are shuttled back to the cytoplasm by transporters Ca2+-binding stabilizing protein 64 Fig 16.29 Generation of glucose from glucose 6-phosphate The stoichiometry of gluconeogenesis: 2 pyruvate + 4ATP + 2GTP + 2NADH + 6H2O glucose + 4ADP + 2GDP + 6Pi + 2NAD+ +2H+ ΔGo’=-48kJ/mol 65 16.4 Gluconeogenesis and Glycolysis Are Reciprocally Regulated Glycolysis is turned on and gluconeogenesis is inhibited 66 Fig 16.30 Reciprocal regulation of gluconeogenesis and glycolysis in the liver The balance between glycolysis and gluconeogenesis in the liver is sensitive to bloodglucose concentration • In the liver, rates of glycolysis and gluconeogenesis are adjusted to maintain blood-glucose levels – signal molecule fructose 2,6-bisphosphate • stimulates phosphofructokinase • inhibits fructose 1,6-bisphosphatase – fructose 2,6-bisphosphate is regulated by • Bifunctional enzyme (a single 55kd polypeptide chain) – Phosphofructokinase 2 (PFK2) » fructose 2,6-bisphosphate is formed – Fructose bisphophatase 2 (FBPase 2) » The hydrolysis of fructose 2,6-bisphosphate » fructose 6-phosphate is formed 67 Glucagon triggers a cyclic AMP signal cascade and activated PKA (also inactive s pyruvate kinase in the liver) 胰高血糖激素 Insulin stimulates the expression of phosphofructokinase, pyruvate kinase and bifunctional enzyme Fig 16.32 Control of the synthesis and degradation of fructose 2,6-bisphosphate 68 Substrate cycles amplify metabolic signals and produce heat • Substrate cycle such as: Glycolysis Gluconeogenesis – Both reactions are not simultaneously fully active in most cells – But some fructose 6-phosphate is phosphorylated to fructose 1,6-bisphosphate during gluconeogenesis – Substrate cycles amplify metabolic signals – Rapid hydrolysis Some times called futile cycle 69 Lactate: Fig 16.35 The cori cycle 1. Lactate can be reverted back to pyruvate and metabolized through TCA cycle 2. The cori cycle Alanine : like lactate, a major precusor of glucose in liver • Generated in muscle • Maintain nitrogen balance 70 71