Carbohydrate metabolism

Section 1Overview

Carbohydrates in general are polyhydroxy aldehydes or ketones or compounds which yield these on hydrolysis.

Biosignificance of CarbohydratesCarbohydrates serve as energy stores (e.g., starch in plants, glycogen in animals), fuels (e.g., glucose), and metabolic intermediates (e.g., ATP, many coenzymes).Carbohydrates serve as structural elements in cell walls of plants (cellulose) or bacteria (peptidoglycans), exoskeletons of arthropods (chitin), and extracellular matrixes of vertebrate animals (proteoglycans).

Carbohydrates are the most abundant biomolecules on earth and have multiple roles in all forms of life.

Carbohydrates serve as recognition signals in glycoproteins and glycolipids determining cell-cell recognition, intracellular location, and metabolic fates of proteins (thus sugars, like nucleic acids and proteins, are also information rich! But codes unknown).Carbohydrates (ribose and deoxyribose) form part of the structural framework of RNA and DNA.

Cells Have Choice among Alternative Substrates, but Glucose Is more Important for Their NeedsThe most important fuctions of carbohydratesGeneration of metabolic energyMaintenance of a normal blood glucose levelSupply of specialized monosaccharides as biosynthetic precursorsSome sugar derivates are important bioactive compounds

Monosacchrides are simple sugars consisting of a single polyhydroxyl aldehyde or ketone unit (e.g., glyceraldehyde, dihydroxyacetone, ribose, glucose, galactose, ribulose, and fructose).Oligosaccharides contain two (disaccharides) or a few monosaccharides joined by glycosidic bonds (e.g., lactose, sucrose, maltose, some covalently linked sugars in glycoproteins and glycolipids).

Carbohydrates can be categorized into monosaccharides, oligosaccharides, and polysacchrides.

Polysaccharides contain long chains of (hundreds to thousands) monosaccharide units joined by glycosidic bonds (e.g., glycogen, starch, cellulose, chitin, and glycosaminoglycans).

Monosacchrides contain one carbonyl group and two or more hydroxyl groups. Monosacchrides can be divided into two families: aldoses and ketoses.Aldoses have their carbonyl groups at the ends of the carbon chains, thus being an aldehyde.Ketoses have their carbonyl groups at places other than the ends, thus being ketones.The simplest aldose is glyceraldehyde, and the simplest ketose is dihyoxyacetone, both being triose.

Monosacchrides containing four, five, and six carbon atoms in their backbones are called tetroses, pentoses (e.g., ribose and deoxyribose), and hexoses (e.g., glucose and fructose), respectively. Hexoses are the most common monosacchrides in nature, including D-glucose, D-mannose, D-galactose, D-fructose.

Glyceraldehyde is conventionally used as the standard for defining D and L configurations: D-glyceraldehyde has the -OH group on the right, L-glyceraldehyde has the -OH group on the left.

For sugars with more than one asymmetric carbon atom, the D- and L- symbols refer to the absolute configuration of the asymmetric carbon farthest to the carbonyl group (e.g., in D-fructose, the -OH on C-5 has the same configuration as the asymmetric carbon in D-glyceraldehyde, therefore, D- and L- glucoses are not enantiomers but stereoisomers!)

The forms of monosaccharides predominate in nature, just as L-amino acids do.

Most of the monosaccharides found in living organisms are the D-isomers (e.g., D-ribose, D-glucose, D-galactose, D-mannose, D-fructose)Each stereoisomer has a different conventional name, ending with -ose suffix.Ketoses are often named by inserting an ul into the name of the corresponding aldoses (e.g., aldopentose is named as ribose, the ketopentose is named as ribulose.

Two sugars differing in configuration at a single asymmetric carbon is called epimers to each other (e.g., D-glucose and D-mannose are epimers at C-2; D-glucose and D-galactose are epimers at C-4).

An aldehyde can react with one alcohol to form a hemiacetal (two alcohol to form acetal), a ketone with an alcohol to form a hemiketal. In the open chain form of glucose, the aldehyde group at C-1 and the hydroxyl group at C-5 react to form two six-membered pyran-like cyclic stereoisomers: the a-D-glucopyranose (the -OH group attached to C-1 locates on a different side from the C-6 atom) and the -D-glucospyranose (-OH of C-1 on the same side of the plane as C-6), thus being specifically called anomers to each other.

Anomeric carbon

Monosaccharide units can link with each other through O-glycosidic bonds to form oligo- and polysaccharides.Disaccharides consist of two monosaccharides linked through an O-glycosidic bond.Sucrose, lactose and maltose are the most abundant disaccharides in nature.In sucrose (common table sugar), the anomeric carbon of one a-D-glucose is joined to the hydroxyl oxygen atom on C-2 of an -D-fructose.

Sucrose, lactose, and maltose can be abbreviated as Glc(1-2)Fru, (or Fru(2-1a)Glc), Gal(1-4)Glc, and Glc(1-4)Glc, respectively.Both lactose and maltose have a free anomeric carbon (not involved in glycosidic bond) that can be oxidized, thus being reducing sugars.The end of an oligo- and polysaccharide having a free anomeric carbon is called the reducing end.Sucrose does not have a reducing end (the anomeric carbons of both saccharide units are involved in glycosidic bond).

The three disaccharides can be hydrolyzed into two monosaccharide units by specific sucrase (also called invertase), lactase (-galactosidase in bacteria), and maltase existing on the outer surface of epithelial cells lining of the small intestines. (milk allergy is due to lack of lactase in the intestines).

Glycogen and starch are mobilizable stores of glucose in animals and plants respectively.Glycogen (mainly in liver and skeleton muscles) is a polymer of (a1-4) linked glucose units with (a1-6) linked branches (occurring about once every 10 glucose residues).Starch can be linear or branched polymers of glucose, called amylose and amylopectin, respectively.Amylose consists of D-glucose residues in (a1-4) linkage.Amylopectin has about one (a1-6) branch per 30 (a1-4) linkages.Amylopectin is like glycogen except for its lower degree of branching.

Each amylose has one nonreducing one and one reducing one, but each amylopectin and glycogen has one reducing end and many nonreducing ends.Starch and glycogen ingested in the diet are hydrolyzed by -amylase (present in saliva and intestinal juice) that break the a1,4 glycosidic linkages between glucose units. (starting from the nonreducing ends). The end of an oligo- and polysaccharide having a free anomeric carbon is called the reducing end.

Cellulose and chitin are structural homopolysaccharides with similar composition and structures.Cellulose, like amylose, is a linear homopolysaccharide of 10,000 or 15,000 D-glucose residues, but with (1-4) linkages.Chitin is a linear homopolysaccharide composed of N-acetyl-D-glucosamine residues also with (1-4) linkages.The only chemical difference between cellulose and chitin is the replacement of a hydroxyl group at C-2 with an acetylated amino group.

Most animals lack enzymes to hydrolyze cellulose but some (like termites and ruminant animals) can use cellulose because of the cellulase secreted by symbiotic microorganisms.The (1-4) linkage allow the polysaccharide chains of cellulose and chitin to take an extended conformation forming parallel fibers through intrachain and interchain hydrogen bond.

Glucose Is the Principal Transported Carbohydrate in humanThe most abundant monosaccharide in dietary carbohydrates is glucose.Glucose Can be transported by bloodGlucose Cannot be stored in cells, only if it can be converted to glycogenBlood glucose level

Glucose Uptake into the Cells Is RegulatedGlucose transporterThe glucose were absorbed by sodium-dependent glucose transporter (SGLT)And then, they were take up by muscle, adipose tissue, brain and other tissue cells through glucose transporter(GLUT)

Glucose transporters (GLUT)GLUT1~5 GLUT1: RBC GLUT4: adipose tissue, muscle

Glucose Oxidation Can Proceed in Many Different PathwayGlucoseglycolysisPyruvateaerobicanaerobic H2O,CO2Lactic acidLactic acid,amino acid,glycerolGlucogenglycogenesispentose phosphate pathway Ribose+NADPH+H+Starch

The metabolism of glucoseglycolysisaerobic oxidationpentose phosphate pathwayglycogen synthesis and catabolismgluconeogenesis

Section 2Glycolysis

GlycolysisThe 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.

1The Development of Biochemistry and the Delineation of Glycolysis Went Hand by Hand1897, Eduard Buchner (Germany), accidental observation : sucrose (as a preservative) was rapidly fermented into alcohol by cell-free yeast extract.The accepted view that fermentation is inextricably tied to living cells (i.e., the vitalistic dogma) was shaken and Biochemistry was born: Metabolism became chemistry!1900s, Arthur Harden and William Young Pi is needed for yeast juice to ferment glucose, a hexose diphosphate (fructose 1,6-bisphosphate) was isolated.

1900s, Arthur Harden and William Young (Great Britain) separated the yeast juice into two fractions: one heat-labile, nondialyzable zymase (enzymes) and the other heat-stable, dialyzable cozymase (metal ions, ATP, ADP, NAD+).1910s-1930s, Gustav Embden and Otto Meyerhof (Germany), studied muscle and its extracts:Reconstructed all the transformation steps from glycogen to lactic acid in vitro; revealed that many reactions of lactic acid (muscle) and alcohol (yeast) fermentations were the same! Discovered that lactic acid is reconverted to carbohydrate in the presence of O2 (gluconeogenesis); observed that some phosphorylated compounds are energy-rich.

(Glycolysis was also known as Embden-Meyerhof pathway).The whole pathway of glycolysis (Glucose to pyruvate) was elucidated by the 1940s.

2The overall glycolytic pathway can be divided into two phasesThe hexose is first phophorylated (thus activated) and then cleaved to produce two three-carbon intermediates at the preparatory phase, consuming ATP.The three-carbon intermediates are then oxidized during the payoff phase, generating ATP and NADH.All intermediates are phosphorylated (as esters or anhydrides) with six (derivatives of Glucose or Fructose) or three carbons (derivatives of dihydroxyacetone, glyceraldehyde, glycerate, or pyruvate).

Ten steps of reactions are involved in the pathway.Only a small fraction (~5%) of the potential energy of the glucose molecule is released and much still remain in the final product of glycolysis, pyruvate.All the enzymes are found in the cytosol (pyruvate will enter mitochondria for further oxidation).

3. The procedure of glycolysisGpyruvatelactic acidglycolytic pathway

1) Glycolytic pathway : G pyruvate including 10 reactions.

Phosphorylated G cannot get out of cell Hexokinase , HK (4 isoenzymes) , glucokinase, GK in liver ;Irreversible The reaction is exergonic.(1) G phosphorylated into glucose 6-phosphate

The first reaction - phosphorylation of glucose

Hexokinase or glucokinase This is a priming reaction - ATP is consumed here in order to get more later ATP makes the phosphorylation of glucose spontaneous

hexokinase glucokinaseoccurrence in all tissues only in liverKm value 0.1mmol/L 10mmol/LSubstrate G, fructose, glucose mannoseRegulation G-6-P InsulinComparison of hexokinase and glucokinase

(2) G-6-P fructose 6-phosphatePhosphohexose isomerase (also called phosphoglucose isomerase) catalyzes the isomerization from glucose 6-P to fructose 6-P, converting an aldose to a ketose.

(3) F-6-P fructose 1,6-bisphosphateThe second phosphorylation Phosphofructokinase-1 (PFK-1, -1) then catalyzes the second phosphorylation step, converting fructose 6-P to fructose 1,6-bisphosphate; the overall rate of glycolysis is mainly controlled at this step; PFK-1 is a highly regulatory enzyme.

(4) F-1,6-BP 2 Triose phosphatesAldolase (), named for the reverse reaction catalyzes the cleavage (lysis) of fructose 1,6-bisphosphate from the middle C-C bond to form two 3-carbon sugars, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate; this is a reversal aldol condensation reaction.

(5) Triose phosphate isomerizationA hydrogen atom is transferred from C-1 to C-3.G2 molecule glyceraldehyde-3-phosphate, consume 2 ATP .

(6) Glyceraldehyde 3-phosphate glycerate 1,3-bisphosphateGlyceraldehyde 3-phosphate dehydrogenase catalyzes first the oxidation and then the phosphorylation of glyceraldehyde 3-P to form glycerate 1,3-bisphosphate, an acyl phosphate (); 2e- are collected by NAD+; a thioester () intermediate is formed between glyceraldehyde 3-P and an essential Cys residue of the enzyme; Pi is used here for the phosphorolysis (); the phosphate group linked to the carboxyl group via a anhydride bond has a high transfer potential.

(7) 1,3-BPG glycerate 3-phosphateThe phosphoglycerate kinase catalyzes the direct transfer of the anhydride phosphate in 1,3-BPG to an ADP to generate an ATP; this is called the substrate-level phosphorylation; 1,3-BPG is a high energy intermediate that leads to ATP formation.

Substrate-Level PhosphorylationATP is formed when an enzyme transfers a phosphate group from a substrate to ADP.Example:PEP to PYR

(8) Glycerate 3-phosphate glycerate 2-phosphate The phosphoglycerate mutase catalyzes the shift of phosphoryl group on 3-phosphoglycerate from C-3 to C-2.

2,3-bisphosphoglycerate is both a coenzyme for the mutase and an intermediate for the reaction; a His residue on the mutase takes phosphoryl group from C-3 of 2,3-BPG and adds it to C-2 of 3-phosphoglycerate, thus forming a phosphorylation cycle; this mutase act in a very similar way as phosphoglucomutase.

(9) Glycerate 2-phosphate phosphoenol pyruvateEnolase () catalyzes the elimination of a H2O from 2-phosphoglycerate to generate phosphoenolglycerate (PEP) with the transfer potential of the phosphoryl group dramatically increased.

(10) PEP pyruvateSecond substrate level phosphorylationIrreversibleThe pyruvate kinase catalyzes the transfer of the phosphoryl group on PEP to ADP to form another molecule of ATP by substrate-level phosphorylation; enolpyruvate is formed and is quickly tautomerized to pyruvate ().

PEP to Pyruvate makes ATP These two ATP (from one glucose) can be viewed as the "payoff" of glycolysis Large, negative G - regulation! This is the third irreversible reaction specific for glycolysis

Under anaerobic conditionsPyruvate Lactate NADH+H+ come from the 6th step of glycolysis (glyceraldehyde-3-phosphate dehydrogenase reaction) .The process from glucose to lactate under anaerobic condition is referred to as anaerobic glycolysis.

2) Pyruvate lactate

Overview of Glycolysis

It is basically an anaerobic processCellular location: cytosol Ten reactions - same in all cells - but rates differ Two phases: A series of reactions in 1st: glucose is broken down to two moleculars of glyceraldehyde-3-phosphate2nd produces two pyruvates, ATPs and NADH Three possible fates for pyruvate

The first phaseDihydroxyacetone phosphate Glyceraldehyde-3-phosphate+

GlycolysisA. Energy Investment Phase:

GrouptransferIsomerizationGrouptransferAldol cleavageThe preparatoryPhase of glycolysisIsomerization

GlycolysisB. Energy Yielding Phase

The payoff phase ofglycolysis DehydrogenationGrouptransfer Group shift DehydrationGrouptransfer

Summary of Glycolysis

Total reaction: C6H12O6 + 2ADP + 2Pi 2CH3CHOHCOOH + 2ATP + 2H2OFormation of ATP: The net yield is 2 ~P or 2 molecules of ATP per glucose.

QuestionWhich of the following enzymes catalyzes a substrate-level phosphorylation reaction? A hexokinase B pyruvate kinase C glyceraldehyde-3-phosphate dehydrogenase D phosphoglycerate kinase E B and DThe substrate level phosphorylation occurs in the process of ?

4. Regulation of GlycolysisThree key enzymes catalyze irreversible reactions : Hexokinase, Phosphofructokinase & Pyruvate Kinase.

1) PFK-1The reaction catalyzed by PFK-1 is usually the rate-limiting step of the Glycolysis pathway. This enzyme is regulated by covalent modification, allosteric regulation.

Regulation of PFK-1ATP is a substrate and an allosteric inhibitor of PFK-1AMP allosterically activates PFK-1 by relieving the ATP inhibition (ADP is also an activator in mammalian systems)Changes in AMP and ADP concentrations can control the flux through PFK-1Elevated levels of citrate (indicate ample substrates for citric acid cycle) also inhibit PFK-1

Regulation of PFK-1 by Fructose 2,6-bisphosphate (F2,6BP)F2,6-BP is a potent activator of PFK-1F2,6-BP is formed from F6P by phosphofructokinase-2 (PFK-2), which is a bi-functional enzymeFormation and hydrolysis of F2,6-BP

bifunctional enzyme

2) Pyruvate kinaseAllosteric 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.

Regulation of Pyruvate Kinase Four PK isozymes exist in mammalian tissuesPK is allosterically activated by F1,6BP, inhibited by ATPGlucagon stimulates protein kinase A which phosphorylates PK converting it to a less active form (liver and intestinal cells) Activator F1,6BP (red) bound with PK

3) Hexokinase and glucokinaseThis enzyme is regulated by covalent modification, allosteric regulation and isoenzyme regulation.Inhibited by its product G-6-P.Insulin induces synthesis of glucokinase.

Regulation of glycolysis 1. When ATP levels are sufficient, glycolysis activity decreasesHexokinase inhibited by excess glucose 6-phosphate PFK-1 is inhibited by ATP and citratePyruvate kinase is inhibited by ATP

2. When ATP is needed, glycolysis is activatedAMP (the product of ATP consumption) relieve the inhibition of PFK-1 by ATPFructose 2,6-bisphosphate (F2,6BP) relieve the inhibition of PFK-1 by ATPPyruvate kinase is activated by F1,6BPStep 1Step 3Step 10

QuestionIn an erythrocyte undergoing glycolysis, what would be the effect of a sudden increase in the concentration of A. ATP? B. AMP? C. fructose-1,6-biophosphate? D. fructose-2,6-biophosphate? E. citrate? F. glucose-6-phosphate?

AnswerIncreased [ATP] or [citrate] inhibits glycolysis.Increased [AMP][fructose-1,6-biophosphate][fructose-2,6-biophosphate],or[glucose-6-phosphate]stimulate glycolysis

The Fate of NADHNADH is energy - two possible fates:

If O2 is available, NADH enters into Mitochondria by two ways, where it is re-oxidized in the electron transport pathway, making ATP in oxidative phosphorylation.

In anaerobic conditions, NADH is re-oxidized by lactate dehydrogenase (LDH), providing additional NAD+ for more glycolysis

Other Substrates for GlycolysisGlycerol, Fructose, mannose and galactose Glycerol is changed into DHAPFructose is primed and cleaved to form dihydroxyacetone phosphate and glyceraldehyde, which are further converted to glyceraldehyde 3-P.Galactose is first converted to Glc-1-P via a UDP-galactose intermediate and UDP-glucose intermediate, then to Glc-6-P.

Triose phosphateisomeraseOne fructose is converted totwo glyceraldehyde 3-P

Glc-P-P-UridineGalactose is converted toglucose 6-P via aUDP-galactose intermediate

Significance of GlycolysisGlycolysis is the emergency energy-yielding pathway. Produce ATPs 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.

The importance of anaerobic glycolysis Only 2ATP(14.6kcal) formed in anaerobic [,n'rubik] glycolysis, whereas the complete oxidation of glucose produces 270kcalHowever, anaerobic glycolysis is usefull Mature erythrocyte need anaerobic glycolysis produce energy (RBC doesnt have mitochondria)When human doing strenuous exercise (ie sprint), skeletal muscle use ATP from anaerobic glycolysisIschemic tissues, nerve etc, use anaerobic glycolysis

SummaryD-glucose is a commonly used as fuel and versatile precursor in almost all organisms.The study of glucose degradation has a rich history in biochemistry (especially for enzymology).Glucose is first converted into two three-carbon pyruvates via the ten-step glycolysis pathway without directly consuming O2 and with a net production of two ATP molecules by substrate-level phosphorylation.Limited amount of energy can be released by oxidizing glucose under anaerobic conditions by fermentation.

The enzymes participating glycolysis may form multiple enzyme complexes, where substrate is channeled from one enzyme to another.The sugar units on glycogen is converted to glucose 1-phosphate via phosphorolysis, which is catalyzed by glycogen phosphorylase.Other monosaccharides are also converted to intermediates of glycolysis for further oxidative degradation.

Phosphofructokinase-1 (PFK-1) is the main point of regulation for controlling the rate of glycolysis.The activity of PFK-1 is regulated by various effectors having various signaling messages of the cell metabolism.Glycolysis and gluconeogenesis is reciprocally regulated to avoid futile cycling of synthesis and degradation.

Mutiple choiceGlycolysis A. takes place in the mitochondrion.B. is the major provider of ATP to muscle during heavy exercise.C. is controlled by levels of fructose-2,6 bis phosphate.D. is the only pathway known from glucose to pyruvate.E. is anaerobic pathway.

Section 3Aerobic Oxidation of Glucose

Fates of PyruvateGo= -146 kJ/molPotential energy = 2840 kJ/molGo= -235 kJ/molGo= -196 kJ/molGo= -1160 kJ/molEthanol fermentation (occurring in yeast and other microorganisms): pyruvate is first decarboxylated and then reduced by NADH, catalyzed by pyruvate decarboxylase and alcohol dehydrogenase respectively.

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.

Under aerobic conditionsUnder aerobic condition, the process which glucose is completely oxidized to CO2 and H2O is called aerobic oxidationThe process take in cytosol and mitochondrion Aerobic oxidation:The first stage----glycolysis, pyruvate is producedThe second stage----pyruvate is oxidized to acetyl-CoAThe third stage---TCA cycle and oxidative phosphorylation

1. Process of aerobic oxidation

GPyrcytosolMitochodriaglycolyticpathwaysecondstagethirdstageCO2+ H2O+ATPPyrCH3CO~SCoAfirststageTAC

1) Oxidative decarboxylation of Pyruvate to Acetyl CoAirreversible;in mitochodria.

Pyruvate dehydrogenase complex: E1 pyruvate dehydrogenaseEs E2 dihydrolipoyl transacetylase E3 dihydrolipoyl dehydrogenase thiamine pyrophosphate, TPP (VB1) HSCoA (pantothenic acid) cofactors lipoic Acid NAD+ (Vpp) FAD (VB2)

HSCoANAD+Pyruvate dehydrogenase complex:

The structure of pyruvate dehydrogenase complex

HSCoA

Arsenic() Compounds Are Poisonous in part because They Sequester Lipoamide

CO2 CoASHNAD+NADH+H+5. NADH+H+1. --TPP 2.3.CoA4.

QuestionWhich is Not the coenzyme of the Pyruvate Dehydrogenase Complex B A. NAD+ B. FMNC. FAD D. TPPE. COA-SH

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.

The TCA Cycle Tricarboxylic acid cyle (TCA cycle), is also called Citric Acid Cycle or Krebs CycleA common metabolic pathway for glucose, amino acid and fatty acidPyruvate from glycolysis is degraded to CO2Some ATP is produced More NADH is made NADH goes on to make more ATP in electron transport and oxidative phosphorylation

(1) Process of reactionsStep 1 The methyl carbon of acety-CoA joins the carbonyl carbon of oxaloacetate via aldol condensation to form citrate ().

Citryl-CoA is a transiently intermediate but hydrolyzed immediately in the active site of citrate synthase; hydrolysis of the thioester bond releases a large amount of free energy, driving the reaction forward; large conformational changes occur after oxaloacetate is bound and after citryl-CoA is formed, preventing the undesirable hydrolysis of acetyl-CoA.

Step 2:Citrate is isomerized into isocitrate (get the six-carbon unit ready for oxidative decarboxylation) via a dehydration step followed by a hydration step; cis-aconitate () is an intermediate during this transformation, thus the catalytic enzyme is named as aconitase, which contains a 4Fe-4S iron-sulfur center directly participating substrate binding and catalysis.

Step 3: Isocitrate is first oxidized and then decarboxylated to form a-ketoglutarate (a-); oxalosuccinate is an intermediate; two electrons are collected by NAD+; the carbon released as CO2 is not from the acetyl group joined; catalyzed by isocitrate dehydrogenase.

Isocitrate Dehydrogenase, IDHOxidative decarboxylation of isocitrate to yield -ketoglutarate Classic NAD+ chemistry (hydride removal) followed by a decarboxylation Isocitrate dehydrogenase is a link to the electron transport pathway because it makes NADH Know the mechanism!

Step 4 a-ketoglutarate undergoes another round of oxidative decarboxylation; decarboxylated first, then oxidized to form succinyl-CoA (A); again the carbon released as CO2 is not from the acetyl group joined; catalyzed by a-ketoglutarate dehydrogenase complex; reactions and enzymes closely resemble pyruvate dehydrogenase complex (with similar E1 and E2, identical E3).

-Ketoglutarate Dehydrogenase ComplexA second oxidative decarboxylation This enzyme is nearly identical to pyruvate dehydrogenase - structurally and mechanistically Five coenzymes used - TPP, CoASH, Lipoic acid, NAD+, FAD You know the mechanism if you remember pyruvate dehydrogenase Another target for arsenic compounds

Step 5 Succinyl-CoA is hydrolyzed to succinate (); the free energy released by hydrolyzing the thioester bond is harvested by a GDP or an ADP to form a GTP or an ATP by substrate-level phosphorylation;

The reversible reaction is catalyzed by succinyl-CoA synthetase (or succinic thiokinase); acyl phosphate and phosphohistidyl enzyme are intermediates; the active site is located at the interface of two subunits; the negative charge of the phospho-His intermediate is stabilized by the electric dipoles of two a helices (one from each subunit).

Succinyl-CoA SynthetaseA substrate-level phosphorylation A nucleoside triphosphateGTP is made Its synthesis is driven by hydrolysis of a CoA ester The mechanism involves a phosphohistidine

Step 6 Succinate is oxidized to fumarate (); catalyzed by a flavoprotein succinate dehydrogenase (with a covalently bound FAD and three iron-sulfur centers), which is tightly bound to the inner membrane of mitochondria; malonate () is a strong competitive inhibitor of the enzyme, that will block the whole cycle.

Succinate DehydrogenaseAn oxidation involving FAD This enzyme is actually part of the electron transport pathway in the inner mitochondrial membrane The electrons transferred from succinate to FAD (to form FADH2) are passed directly to ubiquinone (UQ) in the electron transport pathway

Step 7 Fumarate is hydrated to L-malate by the action of fumarase; the enzyme is highly stereospecific, only act on the trans and L isomers, not on the cis and D isomers (maleate and D-malate);

Step 8 Oxaloacetate is regenerated by the oxidation of L-malate; this reaction is catalyzed by malate dehydrogenase with two electrons collected by NAD+.

Citrate cycle

Summary of Krebs Cycle Reducing equivalents

The net reaction of the TCAC:acetylCoA+3NAD++FAD+GDP+Pi+2H2O 2CO2+3NADH+3H++FADH2+GTP+ HSCoA Irreversible and aerobic reaction The enzymes are located in the mitochondrial matrix.

Anaplerotic reaction of oxaloacetate

The Fate of Carbon in TCAEach cycle, 2 carbon become CO2Its nearly, 1 acetyl-CoA was oxidized during 1 cycleIf lable acetyl-CoA using 14C, you will find carbon of CO2 is from oxaloacetate, not acetyl-CoAMaybe during the cycle, they change C with each otherIntermediate products of the cycle including oxaloacetate are not change. They come from glucose

(2) Bio-significance of TCAC Acts as the final common pathway for the oxidation of carbohydrates, lipids, and proteins. 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 10 ATP.pyruvate acetyl CoA: NADH+H+ 2.5 ATP1 G 2 pyruvate : 2(NADH+H+) 5 or 7ATP 1mol G 30 or 32mol ATP 102.5 2 5 7 30 32

3. The regulation of aerobic oxidationThe 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

(2) Citrate synthaseAllosteric activator: ADPAllosteric inhibitor: NADH, succinyl CoA, citrate, ATP(3) Isocitrate dehydrogenaseAllosteric activator: ADP, Ca2+Allosteric inhibitor: ATP(4) -Ketoglutarate dehydrogenaseSimilar with Pyruvate dehydrogenase complex

Regulation of the TCA cycle

Oxidative phosphorylationTCAC

ATP/ADP inhibit TCAC, Oxidative phosphorylation ATP/ADPpromote TCAC Oxidative phosphorylation

4. Pasteur EffectUnder 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 CO2H2O Pyr cant produce to lactate.

The Pasteur EffectUnder anaerobic conditions the conversion of glucose to pyruvate is much higher than under aerobic conditions (yeast cells produce more ethanol and muscle cells accumulate lactate)The Pasteur Effect is the slowing of glycolysis in the presence of oxygenMore ATP is produced under aerobic conditions than under anaerobic conditions, therefore less glucose is consumed aerobically

Section 4Pentose Phosphate Pathway

Pentose Phosphate PathwayAka:Pentose shunt Hexose monophosphate shunt Phosphogluconate pathway It occurs in the cytosol .Two oxidative processes followed by five non-oxidative steps Operates active in the cytosol of liver and adipose cells

Pentose Phosphate Pathway Pentose phosphate pathway converts glucose to specialized products needed by the cells

Provides NADPH Produces ribose-5-P

GlucoseGlucose-6-PFructose-6-PGlycolysisGlycogenPPP70%30%

1.Oxidative Phase Glucose-6-P Dehydrogenase

Irreversible 1st step - highly regulated (inhibited by NADPH)Gluconolactonase

Uncatalyzed reaction happens too 6-Phosphogluconate Dehydrogenase

An oxidative decarboxylation

Regulatory enzymeThe enzyme is highly specific for NADP+; the Km for NAD+ is 1000 greater than for NADP+.

Oxidative Phase

2. The Nonoxidative Phase

Transketolase

transfer of two-carbon units Transaldolase

transfers a three-carbon unit

5537645463C5 + C5 --> C7 + C3C7 + C3 --> C4 + C6C5 + C4 --> C6 + C3Sum:3C5 --> 2C6 + C3+++++

Non-Oxidative Phase Transketolase: requires TPP Transaldolase

The total reactions 3Glucose 6-phosphate+ 6 NADP+2Frucose 6-phosphate+glyceradehyde-3-phosphate+6NADPH+H++3CO2

3. Regulation of pentose phosphate pathway

Glucose-6-phosphate Dehydrogenase is the rate-limiting enzyme. NADPH/NADP+, inhibit; NADPH/NADP+, activate.

4. Significance of pentose Phosphate pathway1) To supply ribose 5-phosphate for bio-synthesis 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;

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 (mono-oxygenases). In liver this enzyme participates in biotransformation.

Variations on the Pentose Phosphate Pathway 1) More ribose-5-P than NADPH is needed2) Both ribose-5-P and NADPH are needed3) More NADPH than ribose-5-P is needed 4) NADPH and ATP are needed, but ribose-5-P is not

Rapidly dividing cells require more ribose 5- phosphate than NADPH.

The need for NADPH and ribose 5-phosphate is balanced.

More NADPH is needed than ribose 5-phosphate; Fatty acid synthesis in adipose cells.

The cell needs both NADPH and ATP

QuestionWhat are the most important products that cells generate by means of the pentose phosphate pathway? ( )A. lactate and ATP.B. ribose-5-phosphate and NADPH.C. NADP+ and ribose-5-phosphate.D. NADPH and UDP-ribose.E. ribulose-1,5-bisphosphate and NADPH.

Section 5Glycogen Metabolism

*Glycogen Metabolism Glycogen BreakdownGlycogen SynthesisGlycogen Storage Diseases

Glycogen is a polymer of glucose residues linked by (14) glycosidic bonds, mainly (16) glycosidic bonds, at branch points.

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GlycogenGlycogen serves as storage carbohydrate in animals, insects and fungiHeavily branched to allow rapid mobilization of glycogen

*Glycogen Breakdown

Glycogen in cells is first converted to Glc-6-P for oxidative degradationGlycogen Storage Diseases

*Glycogen in cells is first converted to Glc-6-P for oxidative degradationA. Glycogen phosphorylase to cleave a1,4 linkagesC. Phosphoglucomutase to convert to usable formB. Glycogen debranching enzymea(1-4) glycosyl transferase and a(1-6) glucosidase activities -> Glc-1-P and GlcGlc-1-P ----------> Glc-6-PGlycogen + Pi ----------> Glycogen + Glc-1-Pn residuesn-1 residuesGlc-6-phosphatase is required for export from liver as GlcGlycogen Breakdown Requires Three Enzymes

No ATP Consumed!No escapeThe glucose unit at the nonreducing terminal of glycogen is removed as Glc-1-P via phosphorolysis: The (a1- 4) glycosidic bond is attacked by an inorganic phosphate). Catalyzed by glycogen phosphorylase (a tetramer).

*Glycogen PhosphorylaseTetramericglycogenphosphorylase(the b form)

glycogen phosphorylases coenzyme pyridoxal phosphate (PLP, ) derived from vitamin B6) act as a general acid-base catalyst.

PLP acts as a general acid-base in the active site of glycogen phosphorylase

Glycogen phosphorylasereaction mechanism1. Formation of an EPiglycogen ternary complex.

2. Oxonium ion intermediate (I) formation from the -linked terminal glucosyl residue involving acid catalysis by Pi facilitated by proton transfer from PLP.

3. Reaction of Pi with overall retention of configuration around C1 to form -D-Glc-1-phosphate. The glycogen, minus one residue, cycles back to step 1.

Glycogen Debranching EnzymeTwo active sites for two different catalytic activities-a bifunctional enzymeRequired for degradation of almost half of glycogen moleculeMaximal rate of debranching enzyme much slower than that of glycogen phosphorylase

Glycogen Debranching EnzymeActs as an (14)transglycosylase(glycosyl transferase)by transferring an (14) linked trisaccharide unit from a limit branch of glycogen to thenonreducing end ofanother branch.

Also catalyzes the hydrolysis of the (16) bond of the remainingglycosyl residue to yield Glc.

PhosphoglucomutaseMechanism of action is similar to that of phosphoglycerate mutase except Ser carries phosphoryl group here.The phosphglucomutase shifts the phosphoryl group from position C-1 to position C-6 on the glucose unit.

Phosphorylase: key E;The end products: 85% of G-1-P and 15% of free G;There is no the activity of glucose 6-phosphatase (G-6-Pase) in skeletal muscle but in liver.Summary:Glycogen catabolism (glycogenolysis)

The process of glycogenesis occurs in cytosol of liver and skeletal muscle mainly. Glycogen synthesis (Glycogenesis)

Glycogen degradationGlycogensynthesis

Glycogen synthesis initially was thought to occur through a direct reverse of the degradation reaction ----wrong

Leloir discovered in 1949 that one hexose is transformed to another via sugar nucleotide and in 1959 that glycogen is synthesized from UDP-glucose!Sugar nucleotides were found to be the activated forms of sugars participating in biosynthesis of glucose.

Glycogen Synthesis Glucose must be activated into UDP-Glc Notice: ADP-Glc for Starch; GDP-Glc or UDP-Glc for Cellulose ()The primer is required GlycogeninProceed from the reducing end to the non-reducing endGlycogen synthase and Glycogen branching enzyme

UDP-Glc SynthesisUDP-Glucose Pyrophosphorylase converts UTP and Glc-1-P to UDP-Glc and pyrophosphate.

Inorganic pyrophosphatase converts PPi to 2Pi, driving reaction

Use of UDP-Glc makes synthesis reaction favorableUDPGs high-energy status permits it to donate glucosyl units to the growing glycogen chain in a thermodynamically favorable reaction.This is a common strategy for carbohydrate addition.

The overall reaction for the formation of UDPG is highly exergonic.

Glycogen synthase Glycogen is added in a 1,4 linkage from UDP-Glc to the non-reducing end of a glycogen chainThe reaction mechanism is apparently like that of phosphorylase

Glycogen is extended from thenonreducing end using UDP-glucose

Glycogenin Glycogen synthase can only extend a chain- cannot initiate de novo synthesisHow is glycogen synthesis initiated?The first step is attachment of a Glc residue to Tyr194 -OH group of the glycogenin protein by a tyrosine glucosyltransferase. Glycogenin autocatalytically extends the glucan chain by up to 7 additional UDP-Glc-supplied residues, forming a primer for the initiation of glycogen synthesis.Once primer started, glycogen synthase can associate to form ternary complex; upon extension of the chain, glycogenin dissociates from the complex

Glycogen branching by amylo-(1,41,6)-transglycosylase.Transfer of terminalchain segments ofabout 7 glucosyl residues to the C6-OHgroups of Glcresidues on the same or another glycogen chain.Each segment mustcome from a chainof at least 11 residues and the new branch point must be at least 4 residues away from other branch points. This reflects the structure of the catalytic site.New branch at least 4 residues from another branchDonor from a branch at least 11 residues long

Summary:Glycogen synthesis

Glycogen Storage Diseases Affect Primarily Liver and/or Muscle Liver symptoms, most often manifested in infancy or childhood:Hepatomegaly (accumulation of normal or abnormal glycogen and sometimes fat), Hypoglycemia (lack of glycogen degradation)Muscle symptoms more often manifest later in life as muscle mass increases, and are most often weakness, pain and myolysis upon strenuous exercise, and myopathy

Glycogen synthase and glycogen phosphorylase are reciprocally regulated in vertebrates by hormones Phosphorylation and dephosphorylation have opposite effects towards the enzymatic acitivities of these two enzymes.Hormones like epinephrine (acting on muscle cells) or glucagon (acting on liver cells) will activate protein kinase A, which will lead to phosphorylation modification of both the glycogen phosphorylase (thus activating it) and the glycogen synthase (thus inactivating it).

Glycogen synthase and phosphorylaseare reciprocallyregulated by hormones viaphosphorylation-dephosphorylation

Section 6Gluconeogenesis

Gluconeogenesis['lu:ku,ni:u'denisis] The synthesis of the glucose or glycogen from non-carbohydrate sources, namely the amino acids, lactate, propionate, and glycerol.

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Concept: The process of transformation of non-carbohydrates to glucose or glycogen is termed as gluconeogenesis. Materials: lactate, glycerol, pyruvate and glucogenic amino acid.Most fatty acids yield only acetyl-CoA ;Acetyl-CoA (through TCA cycle) cannot provide for net synthesis of sugars )Site: mainly liver, kidney.

1. Gluconeogenic pathwayThe 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

Linkage of biotin to lysine residue in pyruvate carboxylase

pyruvateOAAPEP pyruvate carboxylase ()coenzyme is biotin, mitochondria phosphoenolpyruvate carboxykinase(), mitochondria and cytosolSummarization

gluconeogenesis

2) F-1, 6-BP F-6-P

3) G-6-P G

Glucose-6-Phosphatase Conversion of Glucose-6-P to Glucose Presence of G-6-Pase in endoplasmic reticulum of liver and kidney cells makes gluconeogenesis possible Muscle and brain do not do gluconeogenesis G-6-P is hydrolyzed as it passes into the ER

Glucose-6-phosphatase is localized in the ER

2 lactic acid G consume ATP?

From pyruvateto PEP: twoalternative paths

gluconeogenesis

Summary: Gluconeogenesis Something Borrowed, Something New Seven steps of glycolysis are retained:

Steps 2 and 4-9 Three steps are replaced:

Steps 1, 3, and 10 (the regulated steps!)The new reactions provide for a spontaneous pathway (G negative in the direction of sugar synthesis), and they provide new mechanisms of regulation

Comparison of glycolysis and gluconeogenesis pathways

2. Regulation of gluconeogenesisGlycolysis and gluconeogenesis are regulated by hormones and allosteric effectorsThe substrate cycle

(1) Gluconeogenesis is regulated by allosteric and substrate-level control mechanisms Acetyl-CoA is potent allosteric effector of glycolysis and gluconeogenesis. Acetyl-CoA inhibits the pyruvate dehydrogenase complex (of glycolysis), but activates the pyruvate carboxylase (of gluconeogenesis).Fructose-2,6-bisphosphate (a regulator, not an intermediate) in liver cells, signaling a high blood glucose/glucagon level, activates PFK-1 and inhibits FBPase-1.

(1) Gluconeogenesis is regulated by allosteric and substrate-level control mechanisms AMP inhibits fructose 1,6-bisphosphatase (FBPase-1), but activates phosphofructokinase-1 (PFK-1).Citrate inhibits PFK-1 and activates FBPase-1

Key enzymes of gluconeogenesis PEP carboxykinase Pyr carboxylase Fructose-bisphosphatase Glucose-6-phosphatase

F-2,6-BP activatesPFK-1, but inhibitsFBPase-1

The level of F-2,6-BP is controlled by the relative activity of PFK-2 and FBPase-2, which are located in one polypeptide chain and whose activities are regulated by glucagon-stimulated phosphorylation.

(2) Regulation of gluconeogenesis by substrate cycleSubstrate cycle: The interconversion of two substrates catalyzed by different enzymes for single direction reactions is called substrate cycle. The substrate cycle produces net hydrolysis of ATP or GTP.------futile cycle

Regulation of Gluconeogenesis

3. Significance of gluconeogenesisReplenishment of Glucose by Gluconeogenesis and Maintaining Normal Blood Sugar Level.Replenishment of Liver Glycogen.Regulation of Acid-base Balance.

4Lactic acid (Cori) cycleLactate, 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 acidosisreused lactate

Lactic acid cycle

Summarize for GluconeogenesisWhat is gluconeogenesis?Gluconeogenesis is not merely the reverse of glycolysis.Four reactions are unique to gluconeogenesisHow gluconeogenesis is regulated?Why gluconeogenesis is important?

QuestionsGluconeogenesis is ( )A The formation of glycogenB The formation of starchesC The formation of glucose from noncarbohydratesD The formation of glucose from other carbohydrates

During the gluconeogenisis conversion of pyruvate into glucose in the liver, all of the following are involved EXCEPT ( )A pyruvate carboxylase B phosphoenolpyruvate carboxylase C phosphoenolpyruvate carboxykinase D glucose 6-phosphatase E fructose 1,6-bisphosphatase

Section 7Blood Sugar and Its Regulation

Blood SugarIt means glucose in blood

1. The source and fate of blood sugar

Blood sugar level must be maintained within a limited range to ensure the supply of glucose to brain. The blood glucose concentration is 3.896.11mmol/L normally. Hypoglycemia 3.33~3.89mmol/L Feel dizzy, tired, even comaHyperglycemia 7.22~7.78mmol/L glucosuria

2. Regulation of blood sugar level1insulin for decreasing blood sugar levels.2glucagonfor increasing blood sugar levels.3glucocorticoid: for increasing blood sugar levels.4adrenalinefor increasing blood sugar levels.

3. Abnormal Blood Sugar LevelHyperglycemia: > 7.227.78 mmol/L The renal threshold for glucose: 8.8910.00mmol/L Hypoglycemia: < 3.333.89mmol/L

Pyruvate as a junction point

An abnormally high blood glucose level is calledA. hypoglycemiaB. hyperglycemiaC. ketosisD. glycosis

GlucoseglycolysisPyruvateaerobicanaerobic H2O,CO2Lactic acidLactic acid,amino acid,glycerolGlucogenglycogenesispentose phosphate pathway Ribose+NADPH+H+Starch

Mitochondria is the major site forfuel oxidation to generate ATP.

********************chitinATP**,lisrldihaid] *[pairu:veit] ATPGlucagon **AFA:fatty acid**25*PPP :Pantose phosphate pathway*26*6-*27*****28 nonoxidative phase*****Glucose-1-phosphate****3*Membrane barrier and energy barrierOAAmalate----cross the membrane ;OAA---Asp *10**6*ATP=*