Lecture 25

– Quiz Monday Pentose Phosphate Pathway– This lecture is for WED.– Quiz Friday on TCA cycle– Pyruvate Dehydrogenase Complex (PDC)

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.

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Pyruvate Dehydrogenase Complex (PDC) • In aerobic respiration, NAD+ is recycled by the

electron transport chain.• Also able to utilize energy previously stored as lactate.• Acetyl-CoA is made from pyruvate through oxidative

decarboxylation by a multienzyme complex, pyruvate dehydrogense.

• The general reaction catalyzed:

C-O-

H-C=O

CH3

O

+ NAD+ + CoA-SH

Acetyl-CoA + NADH + CO2

Gº’ = -8 kcal/mol

Pyruvate Dehydrogenase Complex (PDC) • Pyruvate dehydrogenase multienzyme complex (PDC)

consists of three enzymes.• Pyruvate dehydrogenase (E1) form dimers that associate with

E2 at the center of the cubic edges.• Dihydrolipoyl transacetylase (E2) core of the enzyme. In E.

coli has 24 identical subunits with cubic symmetry. • Dihydrolipoyl dehydrogenase (E3) form dimers that are

located on the centers of the cube’s six faces.Gram-negative bacteria have this type. Another type is dodecahedral form found in eukaryotes and

gram-positive bacteria.

Figure 21-4 Structural organization of the E. coli PDC.

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Dihydrolipoyl transacetylase (E2) core

Orange spheres are the 24 pyruvate dehydrogenase (E1) form dimers

Combined a and bDihydrolipoyl transacetylase (E2) core indicated by shaded cube

Purple spheres are the 12 dihydrolipoyl dehydrogenase (E3) subunits also form dimers

Pyruvate Dehydrogenase Complex (PDC) • 5 coenzymes vitaminThiamine pyrophosphate (TPP) thiamineFlavin adenine dinucleotide (FAD) riboflavinCoenzyme A (CoA) pantothenic acidNicotinamide adenine dicleotide (NAD) niacinLipoic acid

• Multienzyme complexes are catalytically efficient and offer advantages over separate enzymes

1. Enzymatic reaction rates are limited by frequency at which enzymes collide with substrates. In a multi-enzyme complex, the distance the substrates must travel is minimized, enhancing rates.

2. Complex formation provides a way of channeling (passing) intermediates between successive enzymes (minimizes side reactions).

3. The reactions may be coordinately controlled.

Figure 21-6 The five reactions of the PDC.

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Pyruvate Dehydrogenase Complex (PDC) • Acetyl-CoA formation occurs over 5 reactions1. Pyruvate dehydrogenase (E1)-decarboxylates pyruvate

using TPP with the intermediate formation of hydroxyethyl-TPP (like pyruvate decarboxylase).

2. Dihydrolipoyl transacetylase (E2)-accepts the hydroxyethyl group from E1.

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.

Mechanism of E1 using TPP

1. Nucleophilic attack by the dipolar cation (ylid) form of TPP on the carbonyl carbon of pyruvate to form a covalent adduct.

2. Loss of carbon dioxide to generate the carbanion adduct in which the thiazolium ring of TPP acts as an electron sink.

3. Pass to next enzyme.

Reaction 1: Pyruvate dehydrogenase (E1) -note how similar to

pyruvate decarboxylase

CH3

P-P-O

S

N

R

(+)

(-)

C-O-

C=O

CH3

O

CH2

CH2

CH3

P-P-O

S

N

R

(+)

CH2

CH2

C-O-

C-OH

CH3

O

CO2TPP (ylid form)

pyruvate

H+

E1

E1

Hydroxyethyl TPP (HETPP)-E1 complex

-CH3

P-P-O

R

CH2

CH2

C-OH

CH3S

N+

Reaction 2: Dihydrolipoyl transacetylase (E2)

S

S

E2

E1

Lipoamide-E2

H+

Hydroxyethyl group carbanion attacks the lipoamide disulfide causing the reduction of the disulfide bond

Hydroxyethyl TPP (HETPP)-E1 complex

CH3

P-P-O

R

CH2

CH2

C-O-H

CH3S

N+

Dihydrolipoyl transacetylase (E2)

HS

S

E2

E1

H+

The TPP is eliminated to form acetyl-dihydrolipoamide and regenerate E1

TPP-E1 complexBack to reaction 1

CH3

P-P-O

R

CH2

CH2

C

CH3

S

N+

Dihydrolipoyl transacetylase (E2)

E1

-

HS

S

E2

Acetyl-dilipoamide-E2

O

C CH3

Reaction 3: Dihydrolipoyl transacetylase (E2)

HS

HS

E2Acetyl-dilipoamide-E2

O

C

CH3

HS

S

E2

O

CoA-SH

CoA-S

dihydrolipamide-E2

E2 catalyzes the transfer of the acetyl group to CoA via a transesterification reaction where the sulfhydryl group of CoA attacks the acetyl group of the acetyl dilipoamide-E2 complex.

+

Reaction 4: Dihydrolipoyl dehydrogenase (E3)

HS

HS

E2E3 is oxidized and catalyzes the oxidation of dihydrolipoamide completing the cycle of E2.

+

FAD

S

SE3 oxidized

FAD

SH

SH

S

S

E2

+E3 reduced

Reaction 5: Dihydrolipoyl dehydrogenase (E3)

E3 is oxidized by the enzyme bound FAD which is reduced to FADH2. This reduces NAD+ to produce NADH.

S

FADH2

S

FAD

SH

SH

NAD+

FAD

S

S

NADH + H+

E3 oxidized

Figure 21-6 The five reactions of the PDC.

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Figure 21-7 Interconversion of lipoamide and dihydrolipoamide.

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Structure of E2

• Consists of several domains• N-terminal Lipoyl domain (80 residues each)-covalently

binds lipoamide• Peripheral subunit-binding domain (35 residues) binds to

E1 and E3• C-terminal catalytic domain (250 residues) catalytic center

and intersubunit binding.• Linked by 20-40 residue Pro/Ala rich segments.

Figure 21-8 Domain structure of the dihydrolipoyl transacetylase (E2) subunit of the PDC.

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The number of lipoyl domains depends on the speciesE. coli, A. vinelandii, n= 3Mammals, n = 2Yeast, n = 1

Figure 21-9 X-Ray structure of a trimer of A. vinelandii dihydrolipoyl transacetylase (E2) catalytic domains. 24

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The N terminal “elbow” extends over neighboring subunit

The CoA and lipoamide bound to enzyme

Structure of E1

• Related to -ketoglutarate dehydrogenase complex and to branched chain keto acid dehydrogenase complex.

• Catalyze the NAD+ linked oxidative decarboxylation of an -keto acid with the transfer of the acyl group to CoA.

• No structure of E1 from PDC has been determined but they make inferences E1 subunits of another keto acid dehydrogenase (P. putida branched-chain-keto acid dehydrogenase, a 2-fold symmetric heterotetramer).

Figure 21-12a X-Ray structure of E1 from P. putida branched-chain -keto acid dehydrogenase. (a)

The 22 heterotetrameric protein.

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Figure 21-12b X-Ray structure of E1 from P. putida branched-chain -keto acid dehydrogenase. (b)

A surface diagram of the active site region.

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Structure of E3

• The reaction is more complex than depicted. • Contains a redox-active disulfide bond that can form a dithiol.• Catalytic mechanism is similar to glutathione reductase.

Figure 21-13a X-Ray structure of dihydrolipoamide dehydrogenase (E3) from P. putida in complex with FAD and NAD+.

(a) The homodimeric enzyme.

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Figure 21-13b X-Ray structure of dihydrolipoamide dehydrogenase (E3) from P. putida in complex with FAD and NAD+.

(b) The enzyme’s active site region.

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Redox active disulfide bridge

Mechanism of E3

• The oxidized enzyme E, which contains the redox-active diulfide bond (S43-S48) binds dihydrolipoamide to form an ES complex.

• His helps with general acid catalysis.• Tyrosine blocks oxidation of FAD by O2 but allows NAD+

access.

•Nucelophillic attack•Proton abstraction yields thiolate ions

Substrate binding

4348

•Substrate thiolate displaces S43 with His as acid catalyst

•S48 forms charge transfer complex with FAD•Lipoamide is released

•Tyr blocks access to FAD.

•NAD+ binds and the Tyr is pushed aside.

•NAD+ is reduced to NADH

•Redox disulfide is reformed.

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•Charge transfer complex-covalent bond formed between Cys48 thiolate and flavin ring. N5 acquires a proton from Cys43.

•Cys43 thiolate nucleophillcially attacks S48 to form the redox active disulfide bond.

•Release of the FADH- anion.

Regulation of PDC

• PDC regulates the entrance of acetyl units derived from carbohydrates into the citric acid cycle.

• The decarboxylation reaction (E1) is irreversible and it is the only pathway for acetyl-CoA synthesis from pyruvate in mammals.

• 2 regulatory systems• Product inhibition by NADH and acetyl-CoA• Covalent modification by phosphorylation/dephosphorylation of the

E1 subunit of pyruvate dehydrogenase.

Figure 21-17a Factors controlling the activity of the PDC.(a) Product inhibition.

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Product inhibition

• NADH and acetyl-CoA compete with NAD+ and CoA for binding sites.

• NADH and acetyl-CoA drive reversible transacetylase (E2) and didhydrolipoyl dehydrogenase (E3) reactions backwards.

Figure 21-17b Factors controlling the activity of the PDC.

(b) Covalent modification in the eukaryotic complex.

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Control by phosporylation/dephosphorylation

• Occurs only in eukaryotic complexes• The E2 subunit has both a pyruvate dehydrogenase

kinase and pyruvate dehydrogenase phosphatase that act to regulate the E1 subunit.

• Kinase inactivates the E1 subunit. Phosphatase activates the subunit.

• Ca2+ is an important secondary messenger, it enhances phosphatase activity.

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Citric acid cycle: 8 enzymes • Oxidize an acetyl group to 2 CO2 molecules and generates 3 NADH, 1 FADH2, and

1 GTP.• Citrate synthase: catalyzes the condensation of acetyl-CoA and oxaloacetate to

yield citrate.• Aconitase: isomerizes citrate to the easily oxidized isocitrate.• Isocitrate dehydrogenase: oxidizes isocitrate to the -keto acid

oxalosuccinate, coupled to NADH formation. Oxalosuccinate is then decarboxylated to form -ketoglutarate. (1st NADH and CO2).

-ketoglutarate dehydrogenase: oxidatively decarboxylates -ketoglutarate to succinyl-CoA. (2nd NADH and CO2).

• Succinyl-CoA synthetase converts succinyl-CoA to succinate. Forms GTP.• Succinate dehydrogenase: catalyzes the oxidation of central single bond of

succinate to a trans double bond, yielding fumarate and FADH2.• Fumarase: catalyzes the hydration of the double bond to produce malate.• Malate dehydrogenase: reforms OAA by oxidizing 2ndary OH group to ketone

(3rd NADH)

Total (PDH and TCA)

3NAD+ + FAD + GDP + Pi + acetyl-CoA

3NADH + FADH2 + GTP + CoA + 2CO2

NAD+ + pyruvate + CoA NADH + acetyl-CoA + CO2

PDH

TCA

Pyruvate4NAD+ FAD GDP + Pi

3CO2

4NADHFADH2

GTP

12ATP

2ATP1ATP

NADH DH

Complex II

Nucleosidediphosphokinase