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Chapter 18: Glycolysis
Often referred to as the Embden – Meyerhof pathway Important because: o For some tissues (brain, kidney medulla, & rapidly contracting skeletal muscle) and cells (erythrocytes & sperm cells), glucose is the only source of metabolic energy o Pyruvate, the final product, is versatile so it can be used in many ways Pyruvate oxidized under aerobic conditions, removing a CO2 and producing Acetyl-CoA, which can undergo metabolism in the TCA cycle and become fully oxidized to synthesize CO2 (HUMANS, contracting muscles) In anaerobic conditions, pyruvate lactate via NADH oxidation (lactic acid fermentation) (MICROORGANISMS – brewer’s yeast) In anaerobic conditions, pyruvate ethanol via NADH oxidation (alcoholic fermentation) Net yield of 2 ATP (2 ATP used in the process in reactions 1 & 3, ATP production in reactions 7 & 10) Net Equation: Glucose + 2 ADP + 2 Pi + NAD+ 2 Pyruvate + 2 ATP + 2 NADH 10 steps, two phases
First Phase 1. Reaction 1: Glucokinase & Hexokinase (IRREVERSIBLE) a. Glucose is phosphorylated by Hexokinase & Glucokinase b. First priming reaction c. Phosphorylation at 6th carbon, activating glucose d. Glucose phosphorylation coupled with ATP hydrolysis to make reaction spontaneous (ΔGo’ = -16.7 kJ/mol after coupling) e. α-D-Glucose + ATP4- α-D-Glucose-6-Phosphate2- + ADP3- + H+ f. Advantage of phosphorylating Glucose: i. A low intracellular glucose concentration is obtained, which favors facilitated diffusion of glucose into the cell ii. Phosphorylation keeps substrate in the cell b/c plasma membrane is impermeable to glucose-6-phosphate (b/c of negative charge) iii. G-6-P is the branch point for several metabolic pathways g. Mg2+ necessary for reaction h. Enzymes: Hexokinase & Glucokinase i. Hexokinase 1. Phosphorylate hexoses like glucose, mannose, and fructose (specificity for D-glucose) 2. 4 isozymes, with type I in brain and mix of types I and II in skeletal muscles
5 mM 3. Organs not highly dependent on glucose b. Isomerization of an aldose to a ketose d. forward and reverse reactions are balanced at 5 mM. Half saturated at much lower concentrations (0. serving as a good glucose buffer but wasting ATP 2.1 mM) than glucokinase 5. Diabetes mellitus patients have low glucokinase 2. Inducible enzyme (controlled by insulin) a. at Vmax 8. Type IV isozymes of hexokinase. found predominately in pancreas & liver a. Carbonyl oxygen shifted from C1 to C2 c. Relatively low affinity for glucose. Enables these organs to glucose sensors and to increase/decrease glucose storage c. Why reaction is necessary: i.1 mM & operates efficiently at a blood glucose level of ~ 4 mM 4. Phosphoglucoisomerase Catalyzes Isomerization of G-6-P b. but easy for primary -OH . Requires a much greater glucose concentration for optimal activity i. Inducible enzyme 7. Prevents cells from extensively phosphorylating glucose when blood glucose concentration is less than 2. At low glucose concentrations. In the liver. Km for glucose is 10 mM – is half saturated at a concentration greater than the normal blood glucose concentration a. permitting CarbonCarbon bond cleavage in fourth (aldolase) reaction ii. Spares generated glucose for other organs. as predicted by Daniel Koshland. Isomerization (carbonyl moved to C2) activates C3. therefore liver doesn’t use glucose it produces b. next step (phosphorylation at C-1) would be tough for hemiacetal -OH. The induced fit model ii. Very weakly inhibited by G-6-P in vivo 4. Binding of glucose (green) induces a conformation change that closes the active site. *Binds glucose and ATP with an Induced Fit a. liver cells minimally phosphorylate glucose via gluconeogenesis i. Allosterically inhibited by Glucose-6-phosphate (one of three regulated reactions) – product inhibition 6. not used in same organ for glycolysis c. Km for glucose is 0. Glucokinase 1.Ramchandani 2 3. At normal blood glucose concentrations. Reaction 2: Phosphoglucoisomerase a.
g. h.= Fructose-6-phosphate2ΔGo’ = 1.+H+ g. proton abstraction leading to enediol formation (step 2). and proton addition to the double bond. Second phosphorylation/priming reaction. f. followed by ring closure (step 3) Requires Mg2+ . 3. Commits cell to glucose metabolism d. e. Fructose-1. Fructose-6-phosphate2. negative ΔG – means PFK is highly regulated .67 kJ/mol i. uses ATP c. Requires Mg2+.Ramchandani 3 iii. Committed step and large. Tetramer w/ four subunits & 4 active sites that can bind to its substrate (Fructose6-phosphate) f. Mechanism for reaction: Figure 18. Reaction 3: Phosphofructokinase (IRREVERSIBLE) a.8 The phosphoglucoisomerase mechanism involves opening of the pyranose ring (step 1). highly specific for G-6-P Enzyme: Phosphoglucoisomerase Glucose-6-phosphate2.+ADP3. Phosphorylation of Fructose-6-Phosphate by Phosphofructokinase b. ΔGo’ = -14.2 kJ/mol i.+ATP4. Enediol intermediate e. Reaction is operating near equilibrium & is readily reversible i.6-biphosphate4.
AMP = reverses allosteric inhibition by ATP 1. vi. Important step for regulation: i. Cleaves Fructose-1. Inhibits fructose-1. At pH = 7 and 37oC. substrate curve 4. PFK decreases activity when energy status is high 5. Restores the hyperbolic dependence of enzyme activity on substrate concentration.6-Biphosphate to create two 3-Carbon Intermediates (Dihydroxyacetone-P & Glyceraldehyde-3-P) b. Reaction 4: Fructose Biphosphate Aldolase a. glycolysis slows down when citric acid cycle reaches saturation v. phosphofructokinase (PFK) behaves cooperatively and the activity plot is sigmoid. Increases phosphofructokinase affinity for its substrate (fructose-6phosphate) 2.6-BP stimulates PFK by decreasing the inhibitory effects of ATP. Cleaves between C3 & C4 = two triose phosphates produced (DHAP & G-3-P) . At high ATP.44 iii. AMP levels can rise dramatically with decrease in ATP b/c of adenylate kinase a.11 F-2. 2 ADP = AMP + ATP. Therefore.6-biphosphate is an allosteric activator 1. Citrate is an allosteric inhibitor 1.Ramchandani 4 ii. β-D-Fructose-2. ii. ATP = high-affinity substrate binding site & low-affinity regulatory site 2. Allosterically inhibited by ATP 1. Keq = 0. ADP = positive effector b/c increases when ATP levels drop iv. Phosphofructokinase couples Glycolysis & Citric Acid Cycle 2. rate of glycolysis activity decreases with high levels of ATP and increases with a greater need for ATP 3. PEP inhibition yields a sigmoidal (S-shaped) velocity vs. Therefore. All four subunits of phosphofructokinase are in the same state: the T ("tense") state or the R ("relaxed") state 4. The binding of PEP to one phosphofructokinase substrate causes a conformation change that affects the ability of the substrate to bind to other subunits 5.6-biphosphatase. reaction is far to the right h. Figure 18. PEP is a feedback inhibitor of phosphofructokinase 2. Phosphofructokinase activity increases with a greater need for ATP (PFK increases activity when energy status is low) 4. the enzyme that catalyzes the reaction in the reverse direction 3. 4. PEP binds to phosphofructokinase at a site other than the active site 3. Phosphoenolpyruvate = product of reaction 9 (PEP) 1.
The chemical evi. The evidence for a Schiff base intermediate for Class I aldolases is described in Problem 18 on page 607 (18) Fructose bisphosphate aldolase in animal muscle is a class I aldolase. ΔGo’ = 23. Dihydroxyacetone-phosphate2. Keq = 10-4 i. Two classes of aldolases found in nature (Cyanobacteria w/ both classes) i. showing the Schiff base as electron sink 4. Animal tissue = class I 1. Unfavorable as written at standard state.ample. which forms a Schiff base intermediate between substrate (for ex.6-biphosphate4. Write a mechanism that explains these observations and provide evidence for the formation of a Schiff base intermediate in the aldolase reaction.12 Mechanism for the Class I aldolase reaction.Ramchandani 5 c.dence for this intermediate comes from studies with aldolase and the reducing agent sodium borohydride. Fructose-1. but cellular ΔG ~ 0 e. fructose-1.9 kJ/mol. NaBH4. Reverse of aldol condensation reaction f.+ Glyceraldehyde3-phosphate2d. Incubation of the enzyme with dihydroxyacetone phosphate and NaBH4 inactivates the enzyme.6bisphosphate or dihydroxyacetone phosphate) and a lysine at the active site (see Figure 18. Interestingly.12). A covalent Schiff base intermediate is formed between the substrate and an active-site lysine 2. no inactivation is observed if NaBH4 is added to the enzyme in the absence of substrate. . Do not require a divalent metal ion 3. Figure 18.
leading to polarization of the substrate carbonyl group.12 (b) In Class II aldolases. an active-site Zn2+ stabilizes the enolate intermediate. Contain an active site metal (Zn2+) 3.Ramchandani 6 ii. . Do not form a covalent E-S intermediate 2. Figure 18. Bacteria & Fungi = Class II 1.
Each glucose has been converted to two molecules of glyceraldehyde-3phosphate. the catalytic residue is Glu165.2 kJ/mol. Keq = 0.see Table 13. Has a turnover number near the diffusion limit i.Ramchandani 7 5. Only G-3-P goes directly into 2nd phase of glycolysis i. Converts DHAP to G-3-P c. This reaction makes it possible for both products of the aldolase reaction to continue in glycolysis b. Triose phosphate isomerase is a near-perfect enzyme . Glu165 in the active site acts as a general base g. Energetically unfavorable but free energy from 2 priming ATP m’cules makes overall Keq constant ~ 1 under standard state conditions . Dihydroxyacetone-Phosphate2. Makes initial C1. f. & C4 carbons respectively d. Glyceraldehyde-3-Phosphate2j. In the yeast enzyme.5 i. h. C5. ΔGo’ = 2. Reaction 5: Triose Phosphate Isomerase a. C2.43 i. & C3 carbons equal to C6. Figure 18.13 A reaction mechanism for triose phosphate isomerase. Involves ene-diol intermediate that can donate either of its hydroxyl protons to a basic residue on the enzyme e.
ΔGo’ = 6. which loses a hydride to NAD+ to become a thioester. . essentially bypassing reaction 7 ii. but reaction’s free energy directed toward the reduction of NAD+ to NADH and formation of a high-energy phosphate compound d. and it is good example of nicotinamide chemistry ii. Phosphorolysis releases 1.14 A mechanism for the glyceraldehyde-3-phosphate dehydrogenase reaction.3-Biphosphoglycerate b.Ramchandani 8 Second Phase 6. Figure 18. This enzyme reaction is the site of action of arsenate (AsO43-) – an anion analogous to phosphate i. but ATP formed in reaction 7 is not made b/c the step has been bypassed 1. The mechanism involves covalent catalysis and a nicotinamide coenzyme.30 kJ/mol i.+ NAD+ 1. Glyceraldehyde-3-P2. Arsenate is an effective substrate for the G3P-DH reaction. which decomposes by hydride (H-) transfer to NAD+ to form a high-energy thioester.3-Biphosphoglycerate4. Reaction of an enzyme sulfhydryl with G3P forms a thiohemiacetal. Oxidation of Glyceraldehyde-3-Phosphate to 1. forming a highly unstable and readily hydrolyzed 1-arseno-3-phosphoglycerate that breaks down to yield 3-phosphoglycerate (produced in reaction 7 by phosphoglycerate kinase).+ NADH + H+ c. and nucleophilic attack by the phosphate displaces the product from the enzyme i.+ Pi2. Glycolysis w/ NO NET ATP! iii. Reaction 6: Glyceraldehyde-3-Phosphate (G-3-P) Dehydrogenase a. Glycolysis continues in the presence of arsenate. Oxidation of an aldehyde to a carboxylic acid = highly exergonic.3-bisphosphoglycerate. Involves nucleophilic attack by Cysteine –SH group on the carbonyl carbon of G3-P to form a hemithioacetal intermediate. Enzyme can be inactivated by reaction w/ iodoacetate e. Uncouples oxidation and phosphorylation events iv.
3-Phosphoglycerate3. Often coupled w/ reaction 6 (seen as a coupled pair) f.3-Biphosphoglycerate4. Sufficiently exergonic at standard state ii. Free energy from this reaction is used to bring previous three closer to equilibrium .6 kJ/mol when coupled w/ reaction 6 i.+ ADP3. 1. ΔGo’ = -12.3-bisphosphoglycerate to ADP to form ATP (“pays off” ATP debt created by priming reactions 1 & 3) b. ADP has been phosphorylated to ATP at the expense of the substrate 2+ c. This is referred to as “substrate-level phosphorylation” i. Mg needed for activity d.Ramchandani 9 7. Reaction 7: Phosphoglycerate Kinase a.+ ATP4e. Transfers a phosphoryl group from 1.
The reaction is actually an intermolecular phosphoryl transfer from C-1 of 1.3-Biphosphoglycerate – and metabolized i.3-BPG from 1.3-BPG (for hemoglobin) is made by circumventing the PGK reaction (Figure 18. An important regulatory molecule is synthesized – 2.3-BPG 1. 2.3-biphosphoglycerate by biphosphoglycerate mutase iii. Stabilizes the deoxy form of hemoglobin & is primarily responsible for the cooperative nature of oxygen binding by hemoglobin vi. 3-phosphoglycerate is then formed by 2.3-BPG v.16 The mutase that forms 2. An important regulator of hemoglobin 1. and undergo intermolecular . Formed from 1.Ramchandani 10 iii. Zelda Rose showed that the enzyme requires a small amount of 2.3-BPG.3-biphosphoglycerate phosphatase 8.3-BPG is carried out by 2.17 A mechanism for the phosphoglycerate mutase reaction in rabbit muscle and in yeast. Transfer phosphate group in 3-phosphoglycerate from C3 to C2 b.3-biphosphoglycerate as a cofactor.3-BPG requires 3-phosphoglycerate. From yeast and rabbit. Rationale for this reaction in glycolysis: It repositions the phosphate to make PEP in the following reaction (enolase) c. 3-Phosphoglycerate3. these enzymes form phosphoenzyme intermediates. Figure 18. 2-Phosphoglycerate3d. ii. Figure 18. use 2. ΔGo’ = 4. Phosphoglycerate mutase enzymes isolated from different sources exhibit different reaction mechanisms f. 2.3-BPG is required to phosphorylate His i.3-BPG to C-2 of 3-phosphoglycerate. Conditions like high ATP and 3-phosphoglycerate levels can reverse reaction g.3-BPG to phosphorylate the His residue before the mechanism can proceed. Hydrolysis of 2. but erythrocytes typically contain 4-5 mM 2.4 kJ/mol e.15) ii. Reaction 8: Phosphoglycerate Mutase a. Most cells contain only a trace of 2. Formation & decomposition of 2.3-bisphosphoglycerate phosphatase iv. Zelda Rose (wife of Nobel laureate Irwin Rose) showed that a bit of 2.
+ H2O c. This phosphoryl group is transferred to the C2 position of the substrate to form a transient. which decomposes by a second phosphoryl transfer from the C3 position of the intermediate to histidine residue on the enzyme iv. the . and H2O iii.Ramchandani 11 phosphoryl group transfers (in which the phosphate in the product is not from 3-phosphoglycerate) iii. Phosphoenolpyruvate3. ΔGo’ = 1. Keq = 0. CO2. Note the phospho-histidine intermediates 1. Figure 18. Intermolecular phosphoryl transfer from C1 of 1. "Energy content" of 2-PG and PEP are similar ii. 2. with a phosphoryl group covalently bound to a histidine residue at the active site 1. The other subunit (b) binds phosphoenol-pyruvate.18 The yeast enolase dimer is asymmetric. 2-phosphoglycerate and PEP with same amount of Potential Metabolic Energy w/ respect to decomposition to Pi. enzyme-bound 2. Prior to her work. Enolase reaction rearranges substrate in a form from which most amount of PE can be released from hydrolysis 1. Reaction 9: Enolase a. the role of the phosphohistidine in this mechanism was not understood v.5 i. Prevalent form of phosphoglycerate mutase is a phosphoenzyme. The active site of one subunit (a) contains 2-phosphoglycerate. Nomenclature note: a “mutase” catalyzes migration of a functional group within a substrate 9. The enolase reaction creates a high-energy phosphate in preparation for ATP synthesis in step 10 of glycolysis.8 kJ/mol. Dehydration by enolase converts 2-phosphoglycerate to Phosphoenolpyruvate (PEP) b.3-Biphosphoglycerate to C2 of 3-Phosphoglycerate g. 2-Phosphoglycerate3. the enolase substrate.3biphosphoglycerate.
Allosterically Inhibited by ATP. NADH i. These two ATP (from one glucose) can be viewed as the "payoff" of glycolysis f. water (yellow). Can be recycled to NAD+ via aerobic or anaerobic conditions 1. This phosphorylated form is more strongly inhibited by ATP & Alanine and has a higher Km for PEP 2. Results in further metabolism of pyruvate . ΔGo’ = -31. Highly favorable and spontaneous conversion of enol tautomer of pyruvate to more stable keto form following phosphoryl group transfer e. Hormones like glucagon activate a cAMP-dependent protein kinase that transfers a phosphoryl group from ATP to the enzyme 1. PEP is a substrate in glucose synthesis via gluconeogenesis h. Li+ (purple).Keq = 3.63 x 105 i.+ ADP3. Phosphoenolpyruvate3. and His159 are shown. Mg2+ (blue). The tautomerization is spontaneous and accounts for much of the free energy change for PEP hydrolysis. 10. Strongly inhibited by F.7 kJ/mol .in the presence of phosphate d. In the presence of physiological levels of PEP. Acetyl CoA & Alanine iii. followed by an enol-keto tautomerization. Liver pyruvate kinase regulated by covalent modifications iv.19 The conversion of phosphoenolpyruvate (PEP) to pyruvate may be viewed as involving two steps: phosphoryl transfer. Requires Mg2+ and is stimulated by K+ and other monovalent cations c. Figure 18.6-biphosphate ii. Mediates transfer of a phosphoryl group from PEP to ADP to make pyruvate and ATP b. this enzyme is inactive g. 11. Allosterically Activated by AMP & fructose-1. Metabolic fates of NADH & Pyruvate? a.+ H+ Pyruvate. Large.+ ATP4d. iv.Ramchandani 12 product of the enolase reaction. Reaction 10: Pyruvate Kinase (IRREVERSIBLE) a. Regulation: i. negative ΔG – indicating that this reaction is subject to regulation ii.
The end products of alcoholic fermentation are thus ethanol and carbon dioxide. Pyruvate reduction reoxidizes NADH ii. which can be seen bubbling to the surface. 2-step process: i. Yeast = reduced to ethanol a. d. Figure 18.21 (b) In oxygen-depleted muscle. Thiamine pyrophosphate (see page 568) is a required cofactor for this enzyme. is catalyzed by alcohol dehydrogenase (Figure 18. Figure 18. Most lactate carried to liver to regenerate glucose via gluconeogenesis 5. Hibernating turtles. Their shells release minerals to buffer the lactate throughout the period of hibernation. the reduction of acetaldehyde to ethanol by NADH. At pH 7. Fermentation: the production of ATP energy by reaction pathways in which organic molecules function as donors and acceptors of electrons a. The second step. NADH from glycolysis & citric acid cycle is oxidized to NAD+ in the mitochondrial ETC iii. NADH is oxidized by lactate dehydrogenase (LDH). Pyruvate i.21). Large amounts of ATP are generated rapidly 4. In anaerobic conditions. A “mash” of corn and other grains is fermented by yeast. If O2 is available (aerobic conditions). c. Pyruvate reduced to lactate by lactate dehydrogenase 3. become “anoxic” and convert glucose mainly to lactate. Aerobic conditions 1. Pyruvate can be sent to the citric acid/TCA cycle w/ the production of additional NADH & FADH2 2. making ATP in oxidative phosphorylation iv. Under aerobic conditions. NADH is oxidized in the electron transport pathway. providing additional NAD+ for more glycolysis b.21 (a) Pyruvate reduction to ethanol in yeast provides a means for regenerating NAD+ consumed in the glyceraldehyde-3-P dehydrogenase reaction. 2. 3. Reduced amount of energy yield from glucose breakdown . (Right) Fermentation at a bourbon distillery. b. producing ethanol and CO2. Other microorganisms & animals = reduced to lactate a. Anaerobic conditions 1.Ramchandani 13 ii. NAD+ is regenerated in the lactate dehydrogenase reaction. ii. Pyruvate is decarboxylated to acetaldehyde by pyruvate decarboxylase in an essentially irreversible reaction. trapped beneath ice and lying in mud. the reaction equilibrium strongly favors ethanol.
forming galactose-1phosphate. phosphofructokinase. PK) – hexokinase. 2) UDP-glucose:galactose-1-phosphate uridylyltransferase converts galactose-1-phosphate to UDP-galactose by transferring UDP from UDP-glucose to galactose-1-phosphate. UDP-glucose is converted to glucose-1-phosphate.Ramchandani 14 How do cells regulate Glycolysis? o Standard state ΔG values are scattered. derived from lactose hydrolysis. negative ΔG These 3 reactions with large negative ΔG are sites of regulation (HK. 3) UDP-galactose 4-epimerase converts UDP-galactose to UDPglucose (which is converted to glucose-1-phosphate in step 2). and pyruvate kinase o Regulation of these three reactions can turn glycolysis off and on o Gluconeogenesis & reverse enzymes for three key reactions Glucose Alternatives o Galactose The galactose derivative that enters the glycolytic pathway is glucose-6phosphate Galactose. with both plus and minus values and no apparent pattern o The plot of ΔG values in cells is revealing: Most values near zero o 3 of 10 reactions have large. PFK. In this reaction. . is converted to glucose-1phosphate 1) Galactokinase phosphorylates galactose.
Glycerol-3-P is then oxidized to dihydroxyacetone phosphate by the action of glycerol phosphate dehydrogenase. It can be converted to glycerol-3-P by glycerol kinase. o The Mannose derivative that enters the glycolytic pathway is fructose-6phosphate Mannose is converted to fructose-6-phosphate. and is converted to fructose-1-phosphate in the liver by the enzyme fructokinase. is converted to fructose6-phosphate in most tissues. 2) Mannose-6-phosphate is isomerized to fructose-6-phosphate.25 The galactose-1-phosphate uridylyltransferase reaction involves a “ping-pong” kinetic mechanism. o Glycerol can also enter glycolysis Glycerol is produced in the decomposition of triacylglycerols. . Fructose-1-phosphate is then cleaved by fructose-1-phoshate aldolase.Ramchandani 15 Figure 18. generating dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. o The fructose derivative that enters the glycolytic pathway in the liver is DHAP & G3P Fructose. 1) Hexokinase catalyzes the phosphorylation of mannose to mannose-6-phosphate. derived from the hydrolysis of sucrose.
Ramchandani 16 .
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