Lecture 24

– Quiz Mon. on Pentose Phosphate Pathway– Glycogen regulation– Quiz next Fri. on TCA cycle

Figure 23-25The pentose phosphate pathway.

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Glycogen biosynthesis

Most important storage form of sugar Glycogen - highly branched (1 per 10) polymer of glucose with (1,4) backbone and (1,6) branch points. More branched than starch so more free ends.

Average molecular weight -several million in liver, muscle.1/3 in liver (more concentrated but less overall mass (5-8%)),2/3 in muscle (1%).

Not found in brain - brain requires free glucose (120 g/ day) supplied in diet or from breakdown of glycogen in the liver.

Glucose levels regulated by several key hormones - insulin, glucagon.

Figure 18-1a Structure of glycogen. (a) Molecular formula.

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Figure 18-1bStructure of glycogen. (b) Schematic diagram illustrating its

branched structure.

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Glycogen is an efficient storage form

G-1-P + UTP Glycogen + UDP + Pi

UDP-glucose

Net: 1 ATP required

Glycogen + Pi

1.1 ATP/38 ATP so, about a 3% loss, therefore it is about 97% efficient for storage of glucose

G-6-P

UDP + ATP UTP + ADP

90% 1,4 residues G-1-P G-6-P

10% 1,6 residues Glycogen glucose

Glycogen biosynthesis

3 enzymes catalyze the steps involved in glycogen synthesis:

UDP-glucose pyrophosphorylaseGlycogen synthaseGlycogen branching enzyme

Glycogen biosynthesis

G-6-PGlucose

F-6-P

G-1-P[G-1,6-P2]

PGI

HKMgATP

MgADP

phosphoglucomutase

G-1-PUTP

PPi

UDP-Glucose Pyrophosphorylase

PPase 2PiThe hydrolysis of pyrophosphate to inorganic phosphate is highly exergonic and is catalyzed by inorganic pyrophosphatase

Figure 18-6 Reaction catalyzed by UDP–glucose pyrophosphorylase.

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UDP-Glucose pyrophosphorylase

Coupling the highly exergonic cleavage of a nucleoside triphosphate to form PPi is a common biosynthetic strategy.

The free energy of the hydrolysis of PPi with the NTP hydrolysis drives the reaction forward.

Glycogen synthase

In this step, the glucosyl unit of UDP-glucose (UDPG) is transferred to the C4-OH group of one of glycogen’s nonreducing ends to form an (1,4) glycosidic bond.

Involves an oxonium ion intermediate (half-chair intermediate)

Each molecule of G1P added to glycogen regenerated needs one molecule of UTP hydrolyzed to UDP and Pi.

UTP is replenished by nucleoside diphosphate kinase

UDP + ATP UTP + ADP

Figure 18-7 Reaction catalyzed by glycogen synthase.

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O

Glycogen synthase

All carbohydrate biosynthesis occurs via UDP-sugars

Can only extend an already (1,4) linked glucan change.

First step is mediated by glycogenin, where glucose is attached to Tyr 194OH group.

The protein dissociates after glycogen reaches a minimum size.

Glycogen branching

Catalyzed by amylo (1,41,6)-transglycosylase (branching enzyme)

Branches are created by the terminal chain segments consisting of 7 glycosyl residues to the C6-OH groups of glucose residues on another chain.

Each transferred segment must be at least 11 residues.

Each new branch point at least 4 residues away from other branch points.

Figure 18-8 The branching of glycogen.

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Glycogen Breakdown

Requires 3 enzymes: 1. Glycogen phosphorylase (phosphorylase) catalyzes

glycogen phosphorylysis (bond cleavage by the substitution of a phosphate group) and yields glucose-1-phosphate (G1P)

2. Glycogen debranching enzyme removes glycogen’s branches, allowing glycogen phosphorylase to complete it’s reactions. It also hydrolyzes a(16)-linked glucosyl units to yield glucose. 92% of glycogen’s glucse residues are converted to G1P and 8% to glucose.

3. Phosphoglucomutase converts G1P to G6P-can either go through glycolysis (muscle cells) or converted to glucose (liver).

Glycogen Phosphorylase

A dimer - 2 identical 842 residue subunits.Catalyzes the controlling step of glycogen breakdown.Regulated by allosteric interactions and covalent modification.Two forms of phosphorylase made by regulationPhosphorylase a- has a phosphoryl group on Ser14 in each

subunit.Phosphorylase b-lacks the phosphoryl groups.Inhibitors: ATP, G6P, glucoseActivator: AMPGlycogen forms a left-handed helix with 6.5 glucose residues

per turn. Structure can accommodate 4-5 sugar residues only.Pyridoxal phosphate is an essential cofactor for

phosphorylase.Converts glucosyl units of glycogen to G1P

Figure 18-2aX-Ray structure of rabbit muscle glycogen phosphorylase. (a) Ribbon diagram of a phosphorylase

b subunit.

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Phosphoglucomutase

Converts G1P to G6P.

Reaction is similar to that of phosphoglycerate mutase

Difference between phosphoglycerate mutase and phosphoglucomutase is the amino acid residue to which the phosphoryl group is attached.

Serine in phosphoglucomutase as opposed to His imidazole N in phosphoglycerate mutase.

G1,6P occasionally dissociates from the enzyme, so catalytic amounts are necessary for activity. This is supplied by the enzyme phosphoglucokinase.

Figure 18-4 The mechanism of action of phosphoglucomutase.

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Glycogen debranching enzyme

(14) transglycosylase (glycosyl transferase) transfers a (14) linked trisaccharide unit from a limit branch to a nonreducing end of another branch.

Forms a new (14) linkage with three more units available for phosphorylase.

The (16) bond linking the remaining linkage is hydrolyzed by the same enzyme to yield glucose.

2 active sites on the same enzyme.

Figure 18-5 Reactions catalyzed by debranching enzyme.

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Regulation of glycogen synthesisBoth synthase & phosphorylase exist in two forms.

Phosphorylated at Ser residues by synthase kinase and phosphorylase kinase

Synthase aNormal form

“active”

Synthase bRequires G6P for activation

“inactive”

OH

OH

OP

OP

ATP

ADP

Pi

Synthase kinasephosphoproteinphosphatase

Regulation of glycogen synthesis

Phosphorylase bNormal form

“inactive”

Phosphorylase aIndependent of energy status

active

OH

OH

OP

OP

ATP

ADP

Pi

phosphorylase kinase

phosphoproteinphosphatase

AMP+, ATP-, G6P-

Ca2+

High [ATP] (related to high G6P) inhibits phosphorylase and stimulates glycogen synthase.

Regulation of glycogen synthesis

Process is also under hormonal controlAdrenaline (epinephrine) can regulate glycogen

synthesis/breakdown by stimulating adenylate cyclase

1. External stimulus Adrenaline Adenylate cyclase

ATP

cAMP + PPi

2. R2C2 [C]2 + [R-AMP]2cAMP dependent protein kinase

cAMP

“inactive” “active”

3a. Glycogen synthase a(active)

Glycogen synthase b (inactive)ATP ADP

[C]2

3b. Inactive phosphorylase kinase

Active phosphorylase kinaseATP ADP

[C]2

ATP ADP

Phosphorylase b(inactive)

Phosphorylase a (active)

Consider the whole systemResting muscle

Glycolytic pathway pyruvate ATP

cAMP

O2 respirationInactive phosphorylase b, active synthase aMuscle lacks G6 Pase, Liver PFK inhibited by ATP unless F2,6P2 present

Upon stressEpinephrine Synthase/phosphorylase kinase

Phosphorylse b Phosphorylse a

Figure 23-25The pentose phosphate pathway.

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Why is the pentose phosphate pathway necessary?

• ATP is the “energy currency” of cells, but cells also need reducing power.

• Endergonic reactions require reducing power and ATP– Fatty acids, cholesterol, photosynthesis

• NADPH and NADH are not interchangeable!– Differ only by a phosphate group at the 2’OH.

Common carrier of (H)

NAD(P) Nicotinamide adenine dinculeotide (phosphate)(oxidized form)

O

N N

NN

O

OHHO

O-

O

O-

O

O

OHHO

CH2-O-P-O-P-CH2 N

C-N-H2

(+)

Pi

NAD+ + 2e- NADH + H+

Common carrier of (H)

NAD(P) Nicotinamide adenine dinculeotide (phosphate)(reduced form)

O

N N

NN

O

OHHO

O-

O

O-

O

O

OHHO

CH2-O-P-O-P-CH2 N

C-N-H2

Pi

H H

NADH + H+ NAD+ + 2e- Eº ‘ = 0.31 volt

Pentose phosphate pathway• NADPH and NADH are not interchangeable!

– Differ only by a phosphate group at the 2’OH. • NADH participates in utilizing the free energy of metabolite oxidation to

synthesize ATP• NADPH utilizes the free energy of metaboite oxidation for biosynthesis• Difference is possible because the dehydrogenase enzymes involved in

oxidative and reductive metabolism exhibit a high degree of specificity toward their respective coenzymes.

• Ratios different: • [NAD+]/[NADH] is near 1000 which favors metabolite oxidation.• [NADP+]/[NADPH] is near 0.1 which favors metabolite reduction.

Why is the pentose phosphate pathway necessary?

• NADPH is generated by oxidation of G6P via the pentose phosphate pathway – hexose monophosphate (HMP) pathway, phosphogluconate pathway.

• Alternate to glycolysis.• Produces ribose-5-phosphate (essential for nucleotide

biosynthesis).

3G6P + 6NADP+ + 3H2O

Overall reaction

6NADPH + 6H+ + 3CO2 + 2F6P + GP

Can be considered in 3 stages

Pentose phosphate pathway Can be divided into three stages

1. Oxidative reactions (1-3) which yield NADPH and ribulose-5-phosphate (Ru5P).

3G6P + 6NADP+ + 3H2O 6NADPH + 6H+ + 3CO2 + 3Ru5P

2. Isomeraization and epimeraztion reactions (4,5)-transform Ru5P to ribose-5-phosphate (R5P) or to xyulose-5-phosphate (Xu5P).

R5P + 2Xu5P3Ru5P

3. C-C bond cleavage and formation reactions (6-8)-convert 2Xu5P and R5P to 2F6P and GAP

2F6P + GAPR5P + 2Xu5P

Oxidative reactions of NADPH production (1-3)

1. Glucose-6-phosphate dehydrogenase (G6PD)-catalyzes the net transfer of a hydride ion to NADP+ from C1 of G6P to form 6-phophoglucono--lactone.

2. 6-phosphoglucolactonase-increases the rate of hydrolysis of 6-phophoglucono--lactone to 6-phosphogluconate.

3. 6-phosphogluconate dehydrogenase catalyzes the oxidative decarboxylation of 6-phosphogluconate, a -hydroxy acid, Ru5P and CO2. (similar to isocitrate dehydrogenase)

Reaction 1: The glucose-6-phosphate dehydrogenase reaction.

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G6PD is strongly inhibited by NADPH

Reaction 2: 6-phosphoglucolactonase

O1

O

-2O3P-O

OH

HOOH

2

3

4

56 6-phosphoglucono--lactone

6-phosphoglucolactonaseMg2+

H2O

C

H-C-OH

HO-C-H

H-C-OH

CH2-OPO32-

H-C-OH

O O-

6-phosphogluconate

Spontaneous reaction sped up by the enzyme

Reaction 3: The phosphogluconate dehydrogenase reaction.

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Summary of 1st stage

• 3 reactions take G6P to Ru5P

– G6P + NADP+ 6-phosphoglucono--lactone + NADPH

– 6-phosphoglucono--lactone 6-phosphogluconate

– 6-phosphogluconate + NADP+ Ru5P + CO2 + NADPH

• Generates 2 molecules of NADPH for each G6P• Ru5P must be converted to R5P or Xu5P for further

use.

2nd stage: isomerization and epimerization

• Ru5P is converted to ribose-5-phosphate (R5P) by ribulose-5-phosphate isomerase

• Ru5P is converted to xyulose-5-phosphate (Xu5P) by ribulose-5-phosphate epimerase

• Occur via enediolate intermediates.• R5P is an essential precursor for nucleotide

biosynthesis.• If more R5P is formed than the cell needs,

converted to F6P and GAP for glycolysis.

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ATPDNARNA

3rd stage: carbon-carbon bond cleavage and formation reactions

• Conversion of three C5 sugars to two C6 sugars and one C3 (GAP)

• Catalyzed by two enzymes, transaldolase and transketolase

• Mechanisms generate a stabilized carbanion which interacts with the electrophilic aldehyde center

Transketolase• Transketolase catalyzes the transfer of C2 unit from Xu5P to R5P

resulting in GAP and sedoheptulose-7-phosphate (S7P).• Reaction involves a covalent adduct intermediate between Xu5P and

TPP.• Has a thiamine pyrophosphate cofactor that stabilizes the carbanion

formed on cleavage of the C2-C3 bond of Xu5P.1. The TPP ylid attacks the carbonyl group of Xu5P (C2)2. C2-C3 bond cleavage results in GAP and enzyme bound 2-(1,2-

dihydroxyethyl)-TPP (resonance stabilized carbanion)3. The C2 carbanion attacks the aldehyde carbon of R5P forming an

S7P-TPP adduct.4. TPP is eliminated yielding S7P and the regenerated enzyme.

Thiamine Pyrophosphate (B1)

Thiazolium ring

CH3

CH2

CH3 CH2CH2O-P-P

H

SN

N

N+

very acidic H since the electrons can delocalize into heteroatoms.

Involved in both oxidative and non-oxidative decarboxylation as a carrier of "active" aldehydes.

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Covalent adduct

Carbanion intermediate

Transketolase

• Similar to pyruvate decarboxylase mechanism.• Septulose-7-phosphate (S7P) is the the substrate

for transaldolase.• In a second reaction, a C2 unit is transferred from

a second molecule of Xu5P to E4P (product of transaldolase reaction) to form a molecule of F6P

Transaldolase• Transfers a C3 unit from S7P to GAP yielding erythrose-

4-phosphate (E4P) and F6P.• Reactions occurs by aldol cleavage.• S7P forms a Schiff base with an -amino group of Lys from

the enzyme and carbonyl group of S7P.• Transaldolase and Class I aldolase share a common

reaction mechanism.• Both enzymes are barrel proteins but differ in where

the Lys that forms the Schiff base is located.

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base with S7P carbonyl group

• A Schiff base-stabilized C3 carbanion is formed in aldol cleavage reaction between C3-C4 yielding E4P.

• The enzyme-bound resonance-stabilized carbanion adds to the carbonyl C of GAP to form F6P.

• The Schiff base hydrolyzes to regenerate the original enzyme and release F6P

Figure 23-31 Summary of carbon skeleton rearrangements in the pentose phosphate

pathway.

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Control of Pentose Phosphate Pathway

1. Principle products are R5P and NADPH.2. Transaldolase and transketolase convert excess R5P

into glycolytic intermediates when NADPH needs are higher than the need for nucleotide biosynthesis.

3. GAP and F6P can be consumed through glycolysis and oxidative phosphorylation.

4. Can also be used for gluconeogenesis to form G6P5. 1 molecule of G6P can be converted via 6 cycles of

PPP and gluconeogenesis to 6 CO2 molecules and generate 12 NADPH molecules.

6. Flux through PPP (rate of NADPH production) is controlled by the glucose-6-phosphate dehydrogense reaction.

7. G6PDH catalyzes the first committed step of the PPP.