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Juhi RamchandaniChem 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 – 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 & Glucokinaseb. First priming reactionc. Phosphorylation at 6th carbon, activating glucosed. 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 pathwaysg. Mg2+ necessary for reactionh. Enzymes: Hexokinase & Glucokinase

i. Hexokinase1. 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

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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 enzyme7. At normal blood glucose concentrations, at Vmax8. *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. Glucokinase1. Type IV isozymes of hexokinase, found predominately in pancreas

& livera. Organs not highly dependent on glucoseb. Enables these organs to glucose sensors and to

increase/decrease glucose storagec. In the liver, forward and reverse reactions are balanced at 5

mM, serving as a good glucose buffer but wasting ATP2. Km for glucose is 10 mM – is half saturated at a concentration

greater than the normal blood glucose concentrationa. Requires a much greater glucose concentration for optimal

activityi. Relatively low affinity for glucose; therefore liver

doesn’t use glucose it producesb. At low glucose concentrations, liver cells minimally

phosphorylate glucose via gluconeogenesisi. Spares generated glucose for other organs; not used

in same organ for glycolysisc. Prevents cells from extensively phosphorylating glucose

when blood glucose concentration is less than 2.5 mM3. Very weakly inhibited by G-6-P in vivo4. Inducible enzyme (controlled by insulin)

a. Diabetes mellitus patients have low glucokinase

2. Reaction 2: Phosphoglucoisomerasea. Phosphoglucoisomerase Catalyzes Isomerization of G-6-Pb. Carbonyl oxygen shifted from C1 to C2c. Isomerization of an aldose to a ketosed. Why reaction is necessary:

i. Isomerization (carbonyl moved to C2) activates C3, permitting Carbon-Carbon bond cleavage in fourth (aldolase) reaction

ii. next step (phosphorylation at C-1) would be tough for hemiacetal -OH, but easy for primary -OH

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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. Requires Mg2+ ; highly specific for G-6-Pf. Enzyme: Phosphoglucoisomeraseg. Glucose-6-phosphate2- = Fructose-6-phosphate2- h. ΔGo’ = 1.67 kJ/mol

i. Reaction is operating near equilibrium & is readily reversiblei. Enediol intermediate

3. Reaction 3: Phosphofructokinase (IRREVERSIBLE)a. Phosphorylation of Fructose-6-Phosphate by Phosphofructokinaseb. Second phosphorylation/priming reaction; uses ATPc. Commits cell to glucose metabolismd. Requires Mg2+;e. Tetramer w/ four subunits & 4 active sites that can bind to its substrate (Fructose-

6-phosphate)f. Fructose-6-phosphate2- +ATP4- Fructose-1,6-biphosphate4- +ADP3- +H+ g. ΔGo’ = -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 37oC, reaction is far to the righth. Important step for regulation:

i. Allosterically inhibited by ATP1. ATP = high-affinity substrate binding site & low-affinity

regulatory site2. 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 high5. 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 = 0.44iii. ADP = positive effector b/c increases when ATP levels dropiv. Citrate is an allosteric inhibitor

1. Phosphofructokinase couples Glycolysis & Citric Acid Cycle2. Therefore, glycolysis slows down when citric acid cycle reaches

saturationv. 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 phosphofructokinase2. PEP binds to phosphofructokinase at a site other than the active

site3. PEP inhibition yields a sigmoidal (S-shaped) velocity vs. substrate

curve4. 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 Aldolasea. 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-biphosphate4- Dihydroxyacetone-phosphate2- + Glyceraldehyde-3-phosphate2-

d. ΔGo’ = 23.9 kJ/mol; Keq = 10-4 i. Unfavorable as written at standard state, but cellular ΔG ~ 0

e. Reverse of aldol condensation reactionf. Two classes of aldolases found in nature (Cyanobacteria w/ both classes)

i. Animal tissue = class I1. A covalent Schiff base intermediate is formed between the

substrate and an active-site lysine2. Do not require a divalent metal ion3. 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-1,6-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 II1. Do not form a covalent E-S intermediate2. 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 Isomerasea. 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-Pc. Makes initial C1, C2, & C3 carbons equal to C6, C5, & C4 carbons respectivelyd. Involves ene-diol intermediate that can donate either of its hydroxyl protons to a

basic residue on the enzymee. Each glucose has been converted to two molecules of glyceraldehyde-3-

phosphate.f. Glu165 in the active site acts as a general baseg. 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 13.5i. Has a turnover number near the diffusion limit

i. Dihydroxyacetone-Phosphate2- Glyceraldehyde-3-Phosphate2- j. ΔGo’ = 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

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Second Phase

6. Reaction 6: Glyceraldehyde-3-Phosphate (G-3-P) Dehydrogenasea. Oxidation of Glyceraldehyde-3-Phosphate to 1,3-Biphosphoglycerateb. Glyceraldehyde-3-P2- + Pi

2- + NAD+ 1,3-Biphosphoglycerate4- + NADH + H+ c. ΔGo’ = 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 G-3-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/ iodoacetatee. This enzyme reaction is the site of action of arsenate (AsO4

3-) – 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 eventsiv. 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 Kinasea. 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 substratec. Mg2+ needed for activityd. 1,3-Biphosphoglycerate4- + ADP3- 3-Phosphoglycerate3- + ATP4- e. Often coupled w/ reaction 6 (seen as a coupled pair)f. ΔGo’ = -12.6 kJ/mol when coupled w/ reaction 6

i. Sufficiently exergonic at standard stateii. Free energy from this reaction is used to bring previous three closer to

equilibrium

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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

phosphataseiv. Most cells contain only a trace of 2,3-BPG, but erythrocytes typically

contain 4-5 mM 2,3-BPGv. 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-BPG1. 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 Mutasea. Transfer phosphate group in 3-phosphoglycerate from C3 to C2b. Rationale for this reaction in glycolysis: It repositions the phosphate to make PEP

in the following reaction (enolase)c. 3-Phosphoglycerate3- 2-Phosphoglycerate3- d. ΔGo’ = 4.4 kJ/mole. Phosphoglycerate mutase enzymes isolated from different sources exhibit

different reaction mechanismsf. Zelda Rose (wife of Nobel laureate Irwin Rose) showed that a bit of 2,3-BPG is

required to phosphorylate Hisi. 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.

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ii. From yeast and rabbit, these enzymes form phosphoenzyme intermediates, use 2,3-biphosphoglycerate as a cofactor, and undergo intermolecular 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 intermediates1. Prior to her work, the role of the phosphohistidine in this

mechanism was not understoodv. 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: Enolasea. Dehydration by enolase converts 2-phosphoglycerate to Phosphoenolpyruvate

(PEP)b. 2-Phosphoglycerate3- Phosphoenolpyruvate3- + H2Oc. ΔGo’ = 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 hydrolysis1. The enolase reaction creates a high-energy phosphate in

preparation for ATP synthesis in step 10 of glycolysis.

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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 product of the enolase reaction. Mg2+ (blue); Li+ (purple); water (yellow), and His159 are shown.

iv. Strongly inhibited by F- in the presence of phosphated.

10. Reaction 10: Pyruvate Kinase (IRREVERSIBLE)a. Mediates transfer of a phosphoryl group from PEP to ADP to make pyruvate and

ATPb. Requires Mg2+ and is stimulated by K+ and other monovalent cationsc. Phosphoenolpyruvate3- + ADP3- + H+ Pyruvate- + ATP4- d. ΔGo’ = -31.7 kJ/mol ;Keq = 3.63 x 105

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 transfere. These two ATP (from one glucose) can be viewed as the "payoff" of glycolysis f. Regulation:

i. Allosterically Activated by AMP & fructose-1,6-biphosphateii. Allosterically Inhibited by ATP, Acetyl CoA & Alanine

iii. Liver pyruvate kinase regulated by covalent modificationsiv. Hormones like glucagon activate a cAMP-dependent protein kinase that

transfers a phosphoryl group from ATP to the enzyme1. This phosphorylated form is more strongly inhibited by ATP &

Alanine and has a higher Km for PEP2. In the presence of physiological levels of PEP, this enzyme is

inactiveg. PEP is a substrate in glucose synthesis via gluconeogenesish. 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?

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a. NADHi. Can be recycled to NAD+ via aerobic or anaerobic conditions

1. Results in further metabolism of pyruvateii. Under aerobic conditions, NADH from glycolysis & citric acid cycle is

oxidized to NAD+ in the mitochondrial ETCiii. If O2 is available (aerobic conditions), NADH is oxidized in the electron

transport pathway, making ATP in oxidative phosphorylationiv. In anaerobic conditions, NADH is oxidized by lactate dehydrogenase

(LDH), providing additional NAD+ for more glycolysisb. Pyruvate

i. Anaerobic conditions1. 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 NADHii. 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 dehydrogenase3. Large amounts of ATP are generated rapidly

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4. Most lactate carried to liver to regenerate glucose via gluconeogenesis

5. Reduced amount of energy yield from glucose breakdown

How do cells regulate Glycolysis?o Standard state ΔG values are scattered, 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, negative ΔG

These 3 reactions with large negative ΔG 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 Alternativeso Galactose

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 UDP-glucose (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.

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 fructose-6-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-6-phosphate

Mannose is converted to fructose-6-phosphate.

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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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