Ramchandani 1 Juhi Ramchandani Chem 441 Test 2 Notes

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 brewers 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 (Go = -16.7 kJ/mol after coupling) e. -D-Glucose + ATP4- -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

Ramchandani 2 3. Km for glucose is 0.1 mM & operates efficiently at a blood glucose level of ~ 4 mM 4. Half saturated at much lower concentrations (0.1 mM) than glucokinase 5. Allosterically inhibited by Glucose-6-phosphate (one of three regulated reactions) product inhibition 6. Inducible enzyme 7. At normal blood glucose concentrations, at Vmax 8. *Binds glucose and ATP with an Induced Fit a. Binding of glucose (green) induces a conformation change that closes the active site, as predicted by Daniel Koshland. The induced fit model ii. Glucokinase 1. Type IV isozymes of hexokinase, found predominately in pancreas & liver a. Organs not highly dependent on glucose b. Enables these organs to glucose sensors and to increase/decrease glucose storage c. In the liver, forward and reverse reactions are balanced at 5 mM, serving as a good glucose buffer but wasting ATP 2. Km for glucose is 10 mM is half saturated at a concentration greater than the normal blood glucose concentration a. Requires a much greater glucose concentration for optimal activity i. Relatively low affinity for glucose; therefore liver doesnt use glucose it produces b. At low glucose concentrations, liver cells minimally phosphorylate glucose via gluconeogenesis i. Spares generated glucose for other organs; not used in same organ for glycolysis c. Prevents cells from extensively phosphorylating glucose when blood glucose concentration is less than 2.5 mM 3. Very weakly inhibited by G-6-P in vivo 4. Inducible enzyme (controlled by insulin) a. Diabetes mellitus patients have low glucokinase 2. Reaction 2: Phosphoglucoisomerase a. Phosphoglucoisomerase Catalyzes Isomerization of G-6-P b. Carbonyl oxygen shifted from C1 to C2 c. Isomerization of an aldose to a ketose d. Why reaction is necessary: i. Isomerization (carbonyl moved to C2) activates C3, permitting CarbonCarbon bond cleavage in fourth (aldolase) reaction ii. next step (phosphorylation at C-1) would be tough for hemiacetal -OH, but easy for primary -OH

Ramchandani 3 iii. Mechanism for reaction: Figure 18.8 The phosphoglucoisomerase mechanism involves opening of the pyranose ring (step 1), proton abstraction leading to enediol formation (step 2), and proton addition to the double bond, followed by ring closure (step 3)

Requires Mg2+ ; highly specific for G-6-P Enzyme: Phosphoglucoisomerase Glucose-6-phosphate2- = Fructose-6-phosphate2Go = 1.67 kJ/mol i. Reaction is operating near equilibrium & is readily reversible i. Enediol intermediate e. f. g. h. 3. Reaction 3: Phosphofructokinase (IRREVERSIBLE) a. Phosphorylation of Fructose-6-Phosphate by Phosphofructokinase b. Second phosphorylation/priming reaction; uses ATP c. Commits cell to glucose metabolism d. Requires Mg2+; e. Tetramer w/ four subunits & 4 active sites that can bind to its substrate (Fructose6-phosphate) f. Fructose-6-phosphate2- +ATP4- Fructose-1,6-biphosphate4- +ADP3- +H+ g. Go = -14.2 kJ/mol i. Committed step and large, negative G means PFK is highly regulated

Ramchandani 4 ii. At pH = 7 and 37oC, reaction is far to the right h. Important step for regulation: i. Allosterically inhibited by ATP 1. ATP = high-affinity substrate binding site & low-affinity regulatory site 2. Therefore, rate of glycolysis activity decreases with high levels of ATP and increases with a greater need for ATP 3. Phosphofructokinase activity increases with a greater need for ATP (PFK increases activity when energy status is low) 4. PFK decreases activity when energy status is high 5. At high ATP, phosphofructokinase (PFK) behaves cooperatively and the activity plot is sigmoid. ii. AMP = reverses allosteric inhibition by ATP 1. AMP levels can rise dramatically with decrease in ATP b/c of adenylate kinase a. 2 ADP = AMP + ATP; Keq = 0.44 iii. ADP = positive effector b/c increases when ATP levels drop iv. Citrate is an allosteric inhibitor 1. Phosphofructokinase couples Glycolysis & Citric Acid Cycle 2. Therefore, glycolysis slows down when citric acid cycle reaches saturation v. -D-Fructose-2,6-biphosphate is an allosteric activator 1. Increases phosphofructokinase affinity for its substrate (fructose-6phosphate) 2. Inhibits fructose-1,6-biphosphatase, the enzyme that catalyzes the reaction in the reverse direction 3. Restores the hyperbolic dependence of enzyme activity on substrate concentration. 4. Figure 18.11 F-2,6-BP stimulates PFK by decreasing the inhibitory effects of ATP. vi. Phosphoenolpyruvate = product of reaction 9 (PEP) 1. PEP is a feedback inhibitor of phosphofructokinase 2. PEP binds to phosphofructokinase at a site other than the active site 3. PEP inhibition yields a sigmoidal (S-shaped) velocity vs. substrate curve 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. All four subunits of phosphofructokinase are in the same state: the T ("tense") state or the R ("relaxed") state 4. Reaction 4: Fructose Biphosphate Aldolase a. Cleaves Fructose-1,6-Biphosphate to create two 3-Carbon Intermediates (Dihydroxyacetone-P & Glyceraldehyde-3-P) b. Cleaves between C3 & C4 = two triose phosphates produced (DHAP & G-3-P)

Ramchandani 5 c. Fructose-1,6-biphosphate4- Dihydroxyacetone-phosphate2- + Glyceraldehyde3-phosphate2d. Go = 23.9 kJ/mol; Keq = 10-4 i. Unfavorable as written at standard state, but cellular G ~ 0 e. Reverse of aldol condensation reaction f. Two classes of aldolases found in nature (Cyanobacteria w/ both classes) i. Animal tissue = class I 1. A covalent Schiff base intermediate is formed between the substrate and an active-site lysine 2. Do not require a divalent metal ion 3. Figure 18.12 Mechanism for the Class I aldolase reaction, showing the Schiff base as electron sink

4. 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, which forms a Schiff base intermediate between substrate (for ex- ample, fructose-1,6bisphosphate or dihydroxyacetone phosphate) and a lysine at the active site (see Figure 18.12). The chemical evi- dence for this intermediate comes from studies with aldolase and the reducing agent sodium borohydride, NaBH4. Incubation of the enzyme with dihydroxyacetone phosphate and NaBH4 inactivates the enzyme. Interestingly, no inactivation is observed if NaBH4 is added to the enzyme in the absence of substrate. Write a mechanism that explains these observations and provide evidence for the formation of a Schiff base intermediate in the aldolase reaction.

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ii. Bacteria & Fungi = Class II 1. Do not form a covalent E-S intermediate 2. Contain an active site metal (Zn2+) 3. Figure 18.12 (b) In Class II aldolases, an active-site Zn2+ stabilizes the enolate intermediate, leading to polarization of the substrate carbonyl group.

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5. Reaction 5: Triose Phosphate Isomerase a. Only G-3-P goes directly into 2nd phase of glycolysis i. This reaction makes it possible for both products of the aldolase reaction to continue in glycolysis b. Converts DHAP to G-3-P c. Makes initial C1, C2, & C3 carbons equal to C6, C5, & C4 carbons respectively d. Involves ene-diol intermediate that can donate either of its hydroxyl protons to a basic residue on the enzyme e. Each glucose has been converted to two molecules of glyceraldehyde-3phosphate. f. Glu165 in the active site acts as a general base g. Figure 18.13 A reaction mechanism for triose phosphate isomerase. In the yeast enzyme, the catalytic residue is Glu165.

h. Triose phosphate isomerase is a near-perfect enzyme - see Table 13.5 i. Has a turnover number near the diffusion limit i. Dihydroxyacetone-Phosphate2- Glyceraldehyde-3-Phosphate2j. Go = 2.2 kJ/mol; Keq = 0.43 i. Energetically unfavorable but free energy from 2 priming ATP mcules makes overall Keq constant ~ 1 under standard state conditions

Ramchandani 8 Second Phase 6. Reaction 6: Glyceraldehyde-3-Phosphate (G-3-P) Dehydrogenase a. Oxidation of Glyceraldehyde-3-Phosphate to 1,3-Biphosphoglycerate b. Glyceraldehyde-3-P2- + Pi2- + NAD+ 1,3-Biphosphoglycerate4- + NADH + H+ c. Go = 6.30 kJ/mol i. Oxidation of an aldehyde to a carboxylic acid = highly exergonic; but reactions free energy directed toward the reduction of NAD+ to NADH and formation of a high-energy phosphate compound d. Involves nucleophilic attack by Cysteine SH group on the carbonyl carbon of G3-P to form a hemithioacetal intermediate, which decomposes by hydride (H-) transfer to NAD+ to form a high-energy thioester, and nucleophilic attack by the phosphate displaces the product from the enzyme i. The mechanism involves covalent catalysis and a nicotinamide coenzyme, and it is good example of nicotinamide chemistry ii. Enzyme can be inactivated by reaction w/ iodoacetate e. This enzyme reaction is the site of action of arsenate (AsO43-) an anion analogous to phosphate i. Arsenate is an effective substrate for the G3P-DH reaction, 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), essentially bypassing reaction 7 ii. Glycolysis continues in the presence of arsenate, but ATP formed in reaction 7 is not made b/c the step has been bypassed 1. Glycolysis w/ NO NET ATP! iii. Uncouples oxidation and phosphorylation events iv. Figure 18.14 A mechanism for the glyceraldehyde-3-phosphate dehydrogenase reaction. Reaction of an enzyme sulfhydryl with G3P forms a thiohemiacetal, which loses a hydride to NAD+ to become a thioester. Phosphorolysis releases 1,3-bisphosphoglycerate.

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7. Reaction 7: Phosphoglycerate Kinase a. Transfers a phosphoryl group from 1,3-bisphosphoglycerate to ADP to form ATP (pays off ATP debt created by priming reactions 1 & 3) b. This is referred to as substrate-level phosphorylation i. ADP has been phosphorylated to ATP at the expense of the substrate 2+ c. Mg needed for activity d. 1,3-Biphosphoglycerate4- + ADP3- 3-Phosphoglycerate3- + ATP4e. Often coupled w/ reaction 6 (seen as a coupled pair) f. Go = -12.6 kJ/mol when coupled w/ reaction 6 i. Sufficiently exergonic at standard state ii. Free energy from this reaction is used to bring previous three closer to equilibrium

Ramchandani 10 iii. Conditions like high ATP and 3-phosphoglycerate levels can reverse reaction g. An important regulatory molecule is synthesized 2,3-Biphosphoglycerate and metabolized i. 2,3-BPG (for hemoglobin) is made by circumventing the PGK reaction (Figure 18.15) ii. Formed from 1,3-biphosphoglycerate by biphosphoglycerate mutase iii. 3-phosphoglycerate is then formed by 2,3-bisphosphoglycerate phosphatase iv. Most cells contain only a trace of 2,3-BPG, but erythrocytes typically contain 4-5 mM 2,3-BPG v. An important regulator of hemoglobin 1. Stabilizes the deoxy form of hemoglobin & is primarily responsible for the cooperative nature of oxygen binding by hemoglobin vi. Formation & decomposition of 2,3-BPG 1. Figure 18.16 The mutase that forms 2,3-BPG from 1,3-BPG requires 3-phosphoglycerate. The reaction is actually an intermolecular phosphoryl transfer from C-1 of 1,3-BPG to C-2 of 3-phosphoglycerate. 2. Hydrolysis of 2,3-BPG is carried out by 2,3-biphosphoglycerate phosphatase

8. Reaction 8: Phosphoglycerate Mutase a. Transfer phosphate group in 3-phosphoglycerate from C3 to C2 b. Rationale for this reaction in glycolysis: It repositions the phosphate to make PEP in the following reaction (enolase) c. 3-Phosphoglycerate3- 2-Phosphoglycerate3d. Go = 4.4 kJ/mol e. Phosphoglycerate mutase enzymes isolated from different sources exhibit different reaction mechanisms f. Zelda Rose (wife of Nobel laureate Irwin Rose) showed that a bit of 2,3-BPG is required to phosphorylate His i. Figure 18.17 A mechanism for the phosphoglycerate mutase reaction in rabbit muscle and in yeast. Zelda Rose showed that the enzyme requires a small amount of 2,3-BPG to phosphorylate the His residue before the mechanism can proceed. ii. From yeast and rabbit, these enzymes form phosphoenzyme intermediates, use 2,3-biphosphoglycerate as a cofactor, and undergo intermolecular

Ramchandani 11 phosphoryl group transfers (in which the phosphate in the product is not from 3-phosphoglycerate) iii. Prevalent form of phosphoglycerate mutase is a phosphoenzyme, with a phosphoryl group covalently bound to a histidine residue at the active site 1. This phosphoryl group is transferred to the C2 position of the substrate to form a transient, enzyme-bound 2,3biphosphoglycerate, which decomposes by a second phosphoryl transfer from the C3 position of the intermediate to histidine residue on the enzyme iv. Note the phospho-histidine intermediates 1. Prior to her work, the role of the phosphohistidine in this mechanism was not understood v. Intermolecular phosphoryl transfer from C1 of 1,3-Biphosphoglycerate to C2 of 3-Phosphoglycerate

g. Nomenclature note: a mutase catalyzes migration of a functional group within a substrate 9. Reaction 9: Enolase a. Dehydration by enolase converts 2-phosphoglycerate to Phosphoenolpyruvate (PEP) b. 2-Phosphoglycerate3- Phosphoenolpyruvate3- + H2O c. Go = 1.8 kJ/mol; Keq = 0.5 i. "Energy content" of 2-PG and PEP are similar ii. 2-phosphoglycerate and PEP with same amount of Potential Metabolic Energy w/ respect to decomposition to Pi, CO2, and H2O iii. Enolase reaction rearranges substrate in a form from which most amount of PE can be released from hydrolysis 1. The enolase reaction creates a high-energy phosphate in preparation for ATP synthesis in step 10 of glycolysis. 2. Figure 18.18 The yeast enolase dimer is asymmetric. The active site of one subunit (a) contains 2-phosphoglycerate, the enolase substrate. The other subunit (b) binds phosphoenol-pyruvate, the

Ramchandani 12 product of the enolase reaction. Mg2+ (blue); Li+ (purple); water (yellow), and His159 are shown. iv. Strongly inhibited by F- in the presence of phosphate d. 10. Reaction 10: Pyruvate Kinase (IRREVERSIBLE) a. Mediates transfer of a phosphoryl group from PEP to ADP to make pyruvate and ATP b. Requires Mg2+ and is stimulated by K+ and other monovalent cations c. Phosphoenolpyruvate3- + ADP3- + H+ Pyruvate- + ATP4d. Go = -31.7 kJ/mol ;Keq = 3.63 x 105 i. Large, negative G indicating that this reaction is subject to regulation ii. Highly favorable and spontaneous conversion of enol tautomer of pyruvate to more stable keto form following phosphoryl group transfer e. These two ATP (from one glucose) can be viewed as the "payoff" of glycolysis f. Regulation: i. Allosterically Activated by AMP & fructose-1,6-biphosphate ii. Allosterically Inhibited by ATP, Acetyl CoA & Alanine iii. Liver pyruvate kinase regulated by covalent modifications iv. Hormones like glucagon activate a cAMP-dependent protein kinase that transfers a phosphoryl group from ATP to the enzyme 1. This phosphorylated form is more strongly inhibited by ATP & Alanine and has a higher Km for PEP 2. In the presence of physiological levels of PEP, this enzyme is inactive g. PEP is a substrate in glucose synthesis via gluconeogenesis h. Figure 18.19 The conversion of phosphoenolpyruvate (PEP) to pyruvate may be viewed as involving two steps: phosphoryl transfer, followed by an enol-keto tautomerization. The tautomerization is spontaneous and accounts for much of the free energy change for PEP hydrolysis.

11. Metabolic fates of NADH & Pyruvate? a. NADH i. Can be recycled to NAD+ via aerobic or anaerobic conditions 1. Results in further metabolism of pyruvate

Ramchandani 13 ii. Under aerobic conditions, NADH from glycolysis & citric acid cycle is oxidized to NAD+ in the mitochondrial ETC iii. If O2 is available (aerobic conditions), NADH is oxidized in the electron transport pathway, making ATP in oxidative phosphorylation iv. In anaerobic conditions, NADH is oxidized by lactate dehydrogenase (LDH), providing additional NAD+ for more glycolysis b. Pyruvate i. Anaerobic conditions 1. Yeast = reduced to ethanol a. Figure 18.21 (a) Pyruvate reduction to ethanol in yeast provides a means for regenerating NAD+ consumed in the glyceraldehyde-3-P dehydrogenase reaction. (Right) Fermentation at a bourbon distillery. A mash of corn and other grains is fermented by yeast, producing ethanol and CO2, which can be seen bubbling to the surface. b. 2-step process: i. Pyruvate is decarboxylated to acetaldehyde by pyruvate decarboxylase in an essentially irreversible reaction. Thiamine pyrophosphate (see page 568) is a required cofactor for this enzyme. ii. The second step, the reduction of acetaldehyde to ethanol by NADH, is catalyzed by alcohol dehydrogenase (Figure 18.21). c. At pH 7, the reaction equilibrium strongly favors ethanol. d. The end products of alcoholic fermentation are thus ethanol and carbon dioxide. 2. Other microorganisms & animals = reduced to lactate a. Figure 18.21 (b) In oxygen-depleted muscle, NAD+ is regenerated in the lactate dehydrogenase reaction. Hibernating turtles, trapped beneath ice and lying in mud, become anoxic and convert glucose mainly to lactate. Their shells release minerals to buffer the lactate throughout the period of hibernation. 3. Fermentation: the production of ATP energy by reaction pathways in which organic molecules function as donors and acceptors of electrons a. Pyruvate reduction reoxidizes NADH ii. Aerobic conditions 1. Pyruvate can be sent to the citric acid/TCA cycle w/ the production of additional NADH & FADH2 2. Pyruvate reduced to lactate by lactate dehydrogenase 3. Large amounts of ATP are generated rapidly 4. Most lactate carried to liver to regenerate glucose via gluconeogenesis 5. Reduced amount of energy yield from glucose breakdown

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How do cells regulate Glycolysis? o Standard state G values are scattered, with both plus and minus values and no apparent pattern o The plot of G values in cells is revealing: Most values near zero o 3 of 10 reactions have large, negative G These 3 reactions with large negative G are sites of regulation (HK, PFK, PK) hexokinase, phosphofructokinase, 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, derived from lactose hydrolysis, is converted to glucose-1phosphate 1) Galactokinase phosphorylates galactose, forming galactose-1phosphate. 2) UDP-glucose:galactose-1-phosphate uridylyltransferase converts galactose-1-phosphate to UDP-galactose by transferring UDP from UDP-glucose to galactose-1-phosphate. In this reaction, UDP-glucose is converted to glucose-1-phosphate. 3) UDP-galactose 4-epimerase converts UDP-galactose to UDPglucose (which is converted to glucose-1-phosphate in step 2).

Ramchandani 15 Figure 18.25 The galactose-1-phosphate uridylyltransferase reaction involves a ping-pong kinetic mechanism.

o The fructose derivative that enters the glycolytic pathway in the liver is DHAP & G3P Fructose, derived from the hydrolysis of sucrose, is converted to fructose6-phosphate in most tissues, and is converted to fructose-1-phosphate in the liver by the enzyme fructokinase. Fructose-1-phosphate is then cleaved by fructose-1-phoshate aldolase, generating dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. o The Mannose derivative that enters the glycolytic pathway is fructose-6phosphate Mannose is converted to fructose-6-phosphate. 1) Hexokinase catalyzes the phosphorylation of mannose to mannose-6-phosphate. 2) Mannose-6-phosphate is isomerized to fructose-6-phosphate. o Glycerol can also enter glycolysis Glycerol is produced in the decomposition of triacylglycerols. It can be converted to glycerol-3-P by glycerol kinase. Glycerol-3-P is then oxidized to dihydroxyacetone phosphate by the action of glycerol phosphate dehydrogenase.

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