Ch 18 Notes - Glycolysis - Biochemistry

Ramchandani 1 Juhi Ramchandani Chem 441 –  441 – Test Test 2 Notes

Chapter 18: Glycolysis  

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Often referred to as the Embden –  Embden – Meyerhof Meyerhof pathway Important because: For some tissues (brain, kidney medulla, & rapidly contracting skeletal muscle) o and cells (erythrocytes & sperm cells), glucose is the only source of metabolic energy Pyruvate, the final product, is versatile so it can b e used in many ways wa ys o  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, p yruvate  lactate via NADH oxidation (lactic acid fermentation) (MICROORGANISMS –   –  brewer’s  brewer’s yeast) In anaerobic conditions,  (MICROORGANISMS  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 P i + 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 th c. Phosphorylation at 6 carbon, activating glucose d. Glucose phosphorylation coupled with ATP hydrolysis to make reaction o spontaneous (Δ (ΔG ’ = -16.7 kJ/mol after coupling) 423+ e. α-D-Glucose + ATP    α-D-Glucose-6-Phosphate + ADP  + 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 2+ g. Mg necessary for reaction h. Enzymes: Hexokinase & Glucokinase i. Hexokinase 1. Phosphorylate hexoses like glucose, mannose, an d 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  doesn’t 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, p ermitting 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)

e. f.  g. h. i.

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Requires Mg ; highly specific for G-6-P Enzyme: Phosphoglucoisomerase 22Glucose-6-phosphate = Fructose-6-phosphate o ΔG ’ = 1.67 kJ/mol i. Reaction is operating near equilibrium & is readily reversible Enediol intermediate

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 2+ d. Requires Mg ; e. Tetramer w/ four subunits & 4 active sites that can bind to its substrate (Fructose6-phosphate) 2443+  f.  Fructose-6-phosphate +ATP    Fructose-1,6-biphosphate +ADP  +H  o g. ΔG ’ = -14.2 kJ/mol i. Committed step and large, negative ΔG – means PFK is highly regulated

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ii. At pH = 7 and 37 C, 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. β-D-Fructose-2,6-biphosphate is an allosteric activator  1. Increases phosphofructokinase affinity for its substrate (fructose-6 phosphate) 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)

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c.  Fructose-1,6-biphosphate   Dihydroxyacetone-phosphate + Glyceraldehyde23-phosphate o -4 d. ΔG ’ = 23.9 kJ/mol; Keq = 10 i. Unfavorable as written at standard state, but cellular ΔG ~ 0 e. Reverse of aldol condensation reaction f. Two classes of aldolases found in nature (C yanobacteria 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 pa ge 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-1,6 bisphosphate 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+ 2. Contain an active site metal (Zn ) 2+ 3. Figure 18.12 (b) In Class II aldolases, an active -site Zn stabilizes the enolate intermediate, leading to polarization of the substrate carbonyl group.

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5. Reaction 5: Triose Phosphate Isomerase nd a. Only G-3-P goes directly into 2 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-3 phosphate. 165 f. Glu in the active site acts as a general base g. Figure 18.13 A reaction mechanism for triose phosphate isomerase. In the yeast 165 enzyme, the catalytic residue is Glu .

h. Triose phosphate isomerase is a near-perfect enzyme - see Table 13.5 i. Has a turnover number near the diffusion limit 22i.  Dihydroxyacetone-Phosphate   Glyceraldehyde-3-Phosphate o  j. ΔG ’ = 2.2 kJ/mol; Keq = 0.43 i. Energetically unfavorable but free energy from 2 priming ATP m’cules 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 22+ 4+ b. Glyceraldehyde-3-P  + P i + NAD   1,3-Biphosphoglycerate + NADH + H  o c. ΔG ’ = 6.30 kJ/mol i. Oxidation of an aldehyde to a carboxylic acid = highly exergonic; but reaction’s 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 3e. This enzyme reaction is the site of action of arsenate (AsO4 ) – 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, bu t 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 4334d. 1,3-Biphosphoglycerate + ADP    3-Phosphoglycerate + ATP  e. Often coupled w/ reaction 6 (seen as a coupled pair) o f. ΔG ’ = -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) 33c. 3-Phosphoglycerate   2-Phosphoglycerate o d. ΔG ’ = 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 2,3 biphosphoglycerate, 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) 33b. 2-Phosphoglycerate   Phosphoenolpyruvate + H 2O o c. ΔG ’ = 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 amoun t 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

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 product of the enolase reaction. Mg (blue); Li (purple); water  159 (yellow), and His 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 2+ +  b. Requires Mg and is stimulated by K  and other monovalent cations 33+ 4c.  Phosphoenolpyruvate + ADP  + H    Pyruvate + ATP  o 5 d. ΔG ’ = -31.7 kJ/mol ;Keq = 3.63 x 10 i. Large, negative ΔG – 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 enz yme 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 glucone ogenesis 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 c ycle 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 etha nol 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? Standard state ΔG values are scattered, with both plus and minus values and no o apparent pattern The plot of  ΔG values in cells is revealing: o  Most values near zero 3 of 10 reactions have large, negative ΔG o  These 3 reactions with large negative ΔG are sites of regulation (HK, PFK, PK) – hexokinase, phosphofructokinase, and pyruvate kinase Regulation of these three reactions can turn glycolysis off and on o Gluconeogenesis & reverse enzymes for three key reactions o Glucose Alternatives Galactose o  The galactose derivative that enters the glycolytic pathway is glucose-6 phosphate  Galactose, derived from lactose hydrolysis, is converted to glucose-1 phosphate 1) Galactokinase phosphorylates galactose, forming galactose-1 phosphate. 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). 

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

The fructose derivative that enters the glycolytic pathwa y 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. The Mannose derivative that enters the glycolytic pathway is fructose-6 phosphate  Mannose is converted to fructose-6-phosphate. 1) Hexokinase catalyzes the phosphorylation of mann ose to mannose-6-phosphate. 2) Mannose-6-phosphate is isomerized to fructose-6-phosphate. 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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