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Chapter 15 Glycolysis and the Catabolism of Hexoses An overview on D-glucose metabolism • The major fuel of most organisms, G'o = –2840 kJ/mole if completely oxidized to CO2 and H2O via the glycolysis pathway, citric acid cycle and oxidative phosphorylation (generating ATP) . • Can also be oxidized to make NADPH and ribose-5-P via the pentose phosphate pathway. • Can be stored in polymer form (glycogen or starch) or be converted to fat for long term storage. • Is also a versatile precursor for carbon skeletons of almost all kinds of biomolecules, including amino acids, nucleotides, fatty acids, coenzymes and other metabolic intermediates. 1. The Development of Biochemistry and the Delineation of Glycolysis Went Hand by Hand • 1897, 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 EmbdenMeyerhof pathway). • The whole pathway of glycolysis (Glucose to pyruvate) was elucidated by the 1940s. 2. The overall glycolytic pathway can be divided into two phases • The 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). • Six types of reactions occur: group transfer (kinase), isomerization (isomerase), aldol cleavage (aldolase), dehydrogenation (dehydrogenase), group shift (mutase), dehydration (dehydratase or enolase). • 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). Group transfer Isomerization Group transfer Aldol cleavage Isomerization The preparatory Phase of glycolysis Dehydrogenation Group transfer Group shift Dehydration Group transfer The payoff phase of glycolysis 3. Ten enzymes catalyze the ten reactions of glycolysis • Hexokinase (also glucokinase in liver) catalyzes the first phosphorylation reaction on the pathway: Mg2+ATP2-, not ATP4- is the actual substrate; binding of glucose induces a profound conformational change on the enzyme; the reaction is exergonic and thus thermodynamically favorable (under standard conditions!). • Phosphohexose isomerase (also called phosphoglucose isomerase) catalyzes the isomerization from glucose 6-P to fructose 6-P, converting an aldose to a ketose. • Phosphofructokinase-1 (PFK-1, 磷酸果糖激酶-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; the plant PFK-1 makes use of PPi, instead of ATP at this step. • Aldolase (醛缩酶), named for the reverse reaction catalyzes the cleavage (“lysis”) of fructose 1,6bisphosphate from the middle C-C bond to form two 3carbon sugars, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate; this is a reversal aldol condensation reaction; thermodynamically very unfavorable under standard conditions. • Triose phosphate isomerase (an extremely efficient enzyme) converts dihydroacetone phosphate to glyceraldehyde 3-phosphate; an intramolecular redox reaction (a hydrogen atom is transferred from C-1 to C-3). • Glyceraldehyde 3-phosphate dehydrogenase catalyzes first the oxidation and then the phosphorylation of glyceraldehyde 3-P to form glycerate 1,3-bisphosphate, an acyl phosphate (酰基 磷酸); 2e- are collected by NAD+; a thioester (硫酯) intermediate is formed between glyceraldehyde 3-P and an essential Cys residue of the enzyme; Pi is used here for the phosphorolysis (磷酸解作用); the phosphate group linked to the carboxyl group via a anhydride bond has a high transfer potential. • The 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. • 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 2,3BPG and adds it to C-2 of 3-phosphoglycerate, thus forming a phosphorylation cycle; this mutase act in a very similar way as phosphoglucomutase. • Enolase (烯醇酶) catalyzes the elimination of a H2O from 2-phosphoglycerate to generate phosphoenolglycerate (PEP) with the transfer potential of the phosphoryl group dramatically increased ( G 0` changed from –17.6 to – 61.9 kJ/mol). • The pyruvate kinase (named for the reverse reaction) 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 (丙酮酸). • A net gain of two ATP, two NADH, two pyruvates are resulted when a glucose molecule is oxidized via the glycolysis pathway: Glucose + 2 ADP + 2Pi + 2NAD+ 2 pyruvate + 2ATP + 2H2O + 2NADH + 2H+ Irreversible in cells Hexokinase Induced fit Glucose An aldose An ketose Reversible The committing step Regulatory ADP One subunit of the tetrameric phosphofructokinase-1 (PFK-1) An aldehyde A ketone 1 4 2 5 The “lysis” 3 step 6 Aldol condensation: The combination of two carbonyl compounds (e.g., an aldehyde and a ketone) to form an aldol (a b-hydroxyl-carbonyl compound). A ketose An aldose C-1 no longer carries a large positive charge: hydride ion leaves readily Proposed action mechanism Of glyceraldehyde 3-P dehydrogenase Iodoacetate 碘醋酸 Phosphorolysis (磷酸解作用) Energy-rich intermediate (thioester) Inactive enzyme Substrate-level phosphorylation For ATP generation Enzyme is named for the reverse reaction A proposed action mechanism for phosphoglycerate mutase 4. Glycolytic enzymes may form multienzyme complexes within cells • When proteins are purified from extracts of broken cells in diluted solutions, noncovalent interactions between proteins could be destroyed. • Kinetic and physical evidences suggest that the enzymes act to catalyzed the ten reactions of glycolysis pathway (as enzymes act in other metabolic pathways) may assemble into multienzyme complexes, where intermediates are directly channeled from one enzyme to another, without entering the aqueous solutions, a phenomenon called substrate channeling. Substrate channeling: growing evidences seem to indicate the formation of multienzyme complexes for enzymes working in one metabolic pathway. 5. Fermentation: pyruvate is converted to lactic acid or ethanol under anaerobic conditions • This occurs to regenerate NAD+ for the glycolysis pathway to continue when O2 lacks. • Lactic acid fermentation (occurring in very active skeleton muscle, some bacteria like lactobacilli): pyruvate is reduced by NADH, catalyzed by lactate dehydrogenase. • The lactate produced in muscle can be converted back to glucose by gluconeogenesis in the liver of vertebrates (via the Cori cycle). • Ethanol fermentation (occurring in yeast and other microorganisms): pyruvate is first decarboxylated and then reduced by NADH, catalyzed by pyruvate decarboxylase and alcohol dehydrogenase respectively. • Thiamine pyrophosphate (TPP, 硫胺焦磷酸, derived from vitamin B1) act as the coenzyme of the decarboxylase: It converts pyruvate to an “active aldehyde” and facilitates C-C bond cleavage adjacent to a carbonyl group (also act in pyruvate and a-ketoglutarate dehydrogenases, transketonases); the carbon between the N and S in the thiazole ring is reactive, where a carbanion (负 碳离子) can be easily generated. Pyruvate is reduced to lactate when O2 lacks in a reaction catalyzed by lactate dehydrogenase Named for the Reverse reaction Pyruvate can be decarboxylated and reduced to form ethanol in some microorganisms Present only in those alcohol fermentative organisms Present in many organisms including human Thiamine pyrophosphate (TPP) contains a reactive thiazole ring where a carbanion can be formed Pyruvate is decarboyxlated With the help of TPP, a coenzyme of pyruvate decarboxylase during ethanol fermentation 6. Glycogen in cells is first converted to Glc-6-P for oxidative degradation • The 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), its coenzyme pyridoxal phosphate (PLP, 磷酸吡哆 醛) derived from vitamin B6) act as a general acidbase catalyst. • Phosphorylase stops working when reaching a terminal residue four away from a branch point. • A bifunctional debranching enzyme (160 kD) removes the (a1 6 ) branches in glycogen: the transferase shifts a block of three glucosyl residues from one outer branch to the other; then the (a1 6) glucosidase activity removes the glucose at the end. • Glc-1-P is then converted to Glc-6-P by the catalysis of phosphoglucomutase,(葡萄糖磷酸变位酶) which uses glucose 1,6-bisphosphate as both a cofactor and an intermediate. • An Ser residue on the enzyme facilitates the phosphorylation cycle (a similar role played by a His residue in the phosphoglycerate mutase). • Glc-6-P is further degraded via the glycolysis pathway (or converted to glucose in liver). No ATP Consumed! Tetrameric glycogen phosphorylase (the b form) No escape Pyridoxal phosphate AMP (allosteric Activator) PLP acts as a general acid-base in the active site of glycogen phosphorylase PLP A bifunctional debranching enzyme aids the phophorylase in degrading glycogen. Glucose 1,6-bisphosphate Ser The phosphglucomutase shifts the phosphoryl group from position C-1 to position C-6 on the glucose unit. 7. Other hexoses are also oxidized via the glycolysis pathway • They are also first primed by phosphorylation (at C-1 or C-6). • Fructose 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 UDPgalactose intermediate and UDP-glucose intermediate, then to Glc-6-P. One fructose is converted to two glyceraldehyde 3-P Triose phosphate isomerase Galactose is converted to glucose 6-P via a UDP-galactose intermediate Glc-P-P-Uridine 8. Dietary poly- and disaccharides are hydrolyzed to monosaccharides in the digestive system • Salivary a-amylase (a-淀粉酶) in the mouth hydrolyzes starch (glycogen) into short polysaccharides or oligosacchrides. • Pancreatic a-amylase (active at low pH) continue act to convert the saccharides to mainly maltoses and dextrins (from amylopectin, 枝链淀粉). • Specific enzymes (e.g., lactase, sucrase, maltase,etc.) on the microvilli of the intestinal epithelial cells finally hydrolyze all disaccharides into monosaccharides. • The monosacchrides are then absorbed at the intestinal microvilli and transported to various tissues for oxidative degradation via the glycolytic pathway. • Adults lacking lactase will have lactose intolerance syndrome: the lactose is converted to toxic compounds in the large intestine by the bacteria there, causing abdominal cramps and diarrhea. 9. Pentose phosphate pathway (戊糖磷 酸途径) converts glucose to specialized products needed by the cells • Glc-6-P is first dehydrogenated by a NADP+containing dehydrogenase to form 6phosphoglucono-d-lactone, which is then hydrolyzed to form 6-phosphogluconate (6-磷酸葡萄糖酸). • 6-phosphogluconate then undergoes a oxidative decarboxylation to form D-ribulose 5-P, generating another molecule of NADPH. • D-ribulose 5-P is then converted to ribose 5-P. • When NADPH is the primary requirement in the cell (as in adipocytes), the pentose phosphates are recycled into Glc-6-P via a series of rearrangements of the carbon skeleton, catalyzed by transketolase ( using TPP) and transaldolase (no cofactor involved). • Six five-carbon sugar phosphates are converted to five six-carbon sugar phosphates. • Much more active in adipose tissue than in muscle. • The reverse of this rearrangement, regeneration of six five-carbon sugar phosphate from five sixcarbon sugar phosphate occurs in the Calvin cycle (for photosynthetic fixation of CO2 in plants). The pentose phosphate pathway Looks familiar? The regeneration of six-carbon Glucose 6-P from five-carbon Ribose 5-P in the Pentose phosphate pathway 核酮糖 5-磷酸 木酮糖 5-磷酸 Ribulose 5-P is first isomerized to form xylulose 5-P to initiate the regeneration of glucose 6-P (this reaction is similar to two reactions in glycolysis, what are they?) TPP helps the twocarbon transferring in transketolase TPP (转酮醇酶) Donor (ketose) Acceptor (aldose) The second reaction catalyzed by transketolase in converting six ribulose 5-P to five Glc 6-P. TPP (转酮醇酶) Donor (ketose) Acceptor (aldose) What is in common between this reaction and that catalyzed by pyruvate decarboxylase? A three-carbon unit is transferred from a ketose to an aldose without being helped by cofactors 景天庚酮糖 赤藓糖 转二羟丙酮酶 Donor (ketose) Acceptor (aldose) 10. The glycogen phosphorylase isozymes in muscle and liver are regulated and differently • The carbohydrate metabolism in muscle and liver serve different physiological roles: oxidative degradation to generate ATP for muscle; maintain a constant blood glucose level for liver (producing and exporting glucose when in demand and importing and storing when in excess. • Two isozymes exist (one in liver and one in muscle), both in two interconvertible forms: the a form is phosphorylated and more active; the b form is dephosphorylated and less active. • Both phosphorylation and dephosphorylation occur (I.e., reversible phosphorylation) and catalyzed by specific phosphorylase b kinase and phosphorylase a phosphatase respectively. • The phosphorylase b kinases in muscle and liver are controlled by two different hormones, epinephrine (肾上腺 素) and glucagon(胰增血糖素) respectively. • High level of AMP binds to and activates the b form of the muscle isozyme, which is blocked by a high level of ATP. • High level of glucose binds to the a form of the liver isozyme, exposing the phosphorylated Ser residues to the action of phosphorylase a phosphatase and converting it to the less active b form. The liver glycogen phosphorylase is regulated by allosteric effector AMP in addition to reversible phosphorylation The a form The b form AMP is a positive regulator Glucose PLP Glycogen The a form of Phosphorylase glycogen phosphorylase a (phosphorylated) AMP Ser14-P The reversible phosphorylations of the glycogen phosphorylase isozymes in liver and muscle are regulated by Different hormones The muscle glycogen phosphorylase is negatively regulated by glucose 11. The rate of glycolysis in mammals is mainly controlled at the step acted by phosphofructokinase-1 (PFK-1) • PFK-1 catalyzes an irreversible exergonic reaction, which commits glucose to the glycolysis pathway (away from the pentose phosphate pathway). • PFK-1 is a complex tetrameric enzyme regulated by multiple intracellular signals (allosteric effectors): ATP, citrate being negative ones; AMP, ADP and fructose 2,6-bisphosphate as positive ones. • A regulated bifunctional enzyme (PFK-2 and FBPase-2) synthesizes (from Fru-6-P) and degrades fructose 2,6-bisphosphate. • A feedforward stimulation: Fru-6-P stimulate the synthesis and inhibits the hydrolysis of Fru-2,6bisphosphate, which in turn stimulates PFK-1. Phosphofructokinase-1 (PFK-1) is regulated by many negative and positive effectors ADP Fructose 1,6bisphosphate 12. Hexokinase and pyruvate kinase also set the pace of glycolysis • These two enzymes also catalyzed irreversible exergonic reactions. • Muscle hexokinase is allosterically inhibited by its reaction product Glc-6-P, which accumulates when PFK-1 is inhibited. • The liver hexokinase (also called hexokinase D or glucokinase) has about 100 X less affinity for glucose than that in muscle and is not inhibited by Glc-6-P: its main role is to convert excess glucose to Glc-6-P for glycogen synthesis. • Pyruvate kinase is allosterically inhibited by ATP,alanine, acetyl-CoA, and long-chain fatty acids. • The catalytic activity of the liver pyruvate kinase isozyme (the L type) is also controlled by reversible phosphorylation. 13. Glycolysis and gluconeogenesis are coordinately regulated to avoid the wasteful “futile cycling” • Gluconeogenesis: The pathway converting simpler precursors (e.g., pyruvate and lactate) to glucose, mainly occurring in the liver of mammals. • Gluconeogenesis uses most of the same enzymes of glycolysis, but the three exergonic irreversible reactions (catalyzed by the three regulatory enzymes) are detoured (bypassed). • The unique enzymes catalyzing the two reversing reactions at one detouring step are reciprocally regulated by common allosteric effectors: fructose 2,6-bisphosphate activates PFK-1 (thus activate glycolysis) and at the same time inhibits fructose bisphosphotase 1 or FBPase-1 (thus inhibit gluconeogenesis). • Enzymes catalyzing the non-common steps of paired catabolic and anabolic pathways are often reciprocally regulated to avoid futile cycling. Summary • D-glucose is a commonly used 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. • Glucose 6-phosphate can also be oxidized to form ribose 5-phosphate and NADPH via the pentose phosphate pathway. • Glycogen phosphorylase is regulated by allosteric effectors and reversible phosphorylation, which is in turn controlled by hormones. • The liver and muscle glycogen phosphorylases are regulated different to meet their physiological roles in mammals. • 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. References • Suarez, R. K., Staples, J.F., Lighton, J. R., and West, T. G. (1997) “Relationships between enzymatic flux capacities and metabolic flux rates: noneequilibrium reactions in muscle glycolysis” Proc. Natl. Acad. Sci. USA, 94:7065-7069. • Barford, D., Hu, S. H., and Jonson, L. N. (1991) “Structural mechanism for glycogen phosphorylase by phosphorylation and AMP” J. Mol. Biol., 218:233260. • Hudson, J. W., Golding, G. B., and Crerar, M. M. (1993) “Evolution of allosteric control in glycogen phosphorylase” J. Mol. Biol., 234:700-721. • Hue, L. and Rider, M. H. (1987) “Role of fructose 2,6bisphosphate in the control of glycolysis in mammalian tissues” Biochem. J., 245:313-323. • Schirmer, T and Evans, P. R. (1990) “Structural basis of the allosteric behavior of phosphofructokinase” Nature, 43:140145. • Sprang, S. R., Withers, S. G., Goldsmith, E. J., Fletterick, R. J., and Madsen, N. B. (1991) “Structural basis for the activation of glycogen phosphorylase b be adenosine monophosphate” Science, 254:1367-1371. Glc-1-P can be converted to UDP-glucuronate and ascorbic acid Supplementary: • Via the UDP-Glucose intermediate. • Consumes NADPH when UDP-glucuronate is further converted to ascorbic acid (vitamin C). • Gulonolatone oxidase is a flavoprotein, which is lacking in human and some other animals (therefore ascorbic acid can not be made by human beings). • UDP-glucuronate is used in glucuronidating nonpolar toxins, drugs or carcinogens, thus making them water-soluble and excretable by a family of detoxifying enzymes. Glc-1-P can be converted to UDPglucuronate and ascorbic acid 古洛糖酸 葡糖醛酸 古洛糖酸内酯 UDP-glucuronate is used in detoxification by glucuronidating nonpolar toxins.