biochem notes.ppt

8/10/2019 biochem notes.ppt

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BCHM221Metabolism (4)

CHAPTER 16

The Citric Acid Cycle

4A. Introduction

4B. Pyruvate Dehydrogenase(PDH) Complex

4C. Citric Acid Cycle (CAC/TCA)

4D. Amphibolic Nature of CAC

4E. Regulation

Cellular Respiration:

Aerobic

Phase of

Catabolismwhere

Nutrients

(sugar,

fatty acids,

amino acids)are Oxidized

to H2O and

CO2.

oxidized,

lose e-

e- (energy) is

conserved as:


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atropos.as.arizona.edu

dinosaurs

land plants

flowers

Photosynthesis by (cyanobacteria)glycolysis

cellular respiration


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The matrix contains pyruvate dehydrogenase, enzymesof the CAC,andother pathways, i.e. fatty acid oxidation& amino acid metabolism.

The outer membrane contains large channels and isleaky to ions & small molecules

matrix

innermembrane

outermembrane

inter-membrane

space

mitochondrion

cristae

i) Localization

Glycolysis happens inthe cells cytosol

Pyruvate enters themitochondrion to bemetabolized furtherby the CAC

ii) Mitochondrial

Compartments

4A. CAC Introduction


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Inner membrane infoldings, called cristae, contain parts ofthe respiratory chain & F1FOATP Synthase and is the majorpermeability barrier

It contains various transporters, including a carrier proteinthat allows pyruvate to enter the matrix

ii) MitochondrialCompartments

matrix

innermembrane

outermembrane

inter-membrane

space

mitochondrion

cristae

4A. CAC Introduction


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Electron Micrographof PDH complex from E. coli

4-10 million Da!!!For an enzyme, this is huge!

d = 450

A multienzyme complex: Aseries of intermediates remainbound to the enz molecules.

Five cofactors (4 vit.).

Complex, well coordinatedregulation

4B. PDH Complex


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Mammaliani) Core complex:60 copies of E2(red)

ii) Periphery of complex:

30 copies of E1(tetramerwith subunits 22purple)

12 copies of E3 (a

homodimer - yellow)E1 = Pyruvate DehydrogenaseE2 = Dihydrolipoyl TransacetylaseE3 = Dihydrolipoyl Dehydrogenase


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Voet and Voet Figure 19-6

Full general mechanism of PDH complex from Voet text:

Note: Your text (Fig 16-6) is missing a step that shows the

role of cystine to regenerate the lipoamide


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Derivative of thiamine (vitamin B1).Nutritional deficiency of thiamineberiberi (p. 620)Especially affects the brain, why?

Figure 14-13

Prosthetic Group #1

TPP, The electron sink

Relativelyacidic


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TPP and

pyruvate

decarboxylation

in fermentation.

Pyruvate to

acetaldehyde

by pyruvatedecarboxylase


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Mechanism of TPP

(The electron sink)

The first step in thePDH complex is thesame as the pyruvatedecarboxylase reaction.H+dissociates from the C

between Nand Sto yield acarbanion

The electron-deficient ketocarbon of pyruvate isattacked by the carbanion

This is followed by arearrangement andresonance stabilizationwith the loss of CO2

Enter lipoamide


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(1) e-(H atom) carrier

(2) Acyl carrierLong flexible arm links lipoamideto E

2, the core of the complex,

allowing dithiol of lipoamide toswing from one active site toanother

2 e-+ 2 H+


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CoASH

(Vit. B5)

CoA as an Acyl Carrier

(Gohydrolysis = 31.4 kJ/mol)

(activated for group transfer)


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1. Pyruvate reacts with TPP and is decarboxylated (E1)

3. Lipoamide delivers an acetyl group to CoASH to produceAcetyl-CoA and lipoamide dithiol (E2)

2. Hydroxyethyl TPP reacts with lipoamide (E1/E2)

4. Lipoamide dithiolis reoxidized by a cystine of E3 (E2/E3)to give two cysteine residues

5. FAD regenerates the cystine to produce FADH2(E3)

6. NAD+reacts with FADH2to regenerate FAD and produceNADH (diffuses to deliver electrons to the E.T.C.)

Summary of PDH Reaction Steps

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/krebs.htm#animat1


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Overall

4B. PDH Complex

Acetyl group generated from pyruvate is transferredto coenzyme A

AcetylCoA is central in metabolismit can easilydonate acetate based on its high energy thioester

linkageinput to CAC (acetateCO2)

donates acetate to fatty acid, ketone body, &cholesterol synthesis

The final electron acceptor is NAD+

NADH


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4C. TheCitric Acid

Cycle

Which enzymesmay be

importantfor regulation?

Review

Box 16-2EnzymeNomenclature


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1. Citrate Synthase: Aldol condensation reaction

Citroyl-CoA is a transient intermediate OA causes an induced fit of acetylCoA to the enzyme CoASH can be recycled for PDH What might make this reaction so exergonic? inhibited by high ratios of ATP:ADP, acetyl-CoA:CoA, and

NADH:NAD, as high conc of ATP, acetyl-CoA, and NADH

show that the energy supply is high for the cell.

Inhibited by succinyl-CoA and citrate, examples of product inhibition


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Mechanism: p. 623.


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2. Aconitase: (aconitate hydratase)reversible hydration

Isocitrate is quickly consumed in the cell The reaction is spontaneous despite the +ve G Contains an Fe-S cluster that aids the reaction

and binds substrate


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3. Isocitrate dehydrogenase: oxidative decarboxylation

Two forms of the enzyme (NAD+or NADP+) - isoenzymes Isoform associated with NADP+also found in cytosol

likely to produce NADPH for reductive anabolism


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4. -ketoglutarate dehydrogenase: oxidative decarboxylation

Succinyl CoA thioester conserves the oxidation energy Similar to PDHthree enzymes (E1-TPP, E2-lipoate, E3-FAD)

likely sharing a common protein ancestor CoASH is the succinyl carrier, NAD+accepts electrons

Given the PDH mechanism can you come up with amechanism for this enzyme?


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5. Succinyl-CoA synthetase: synthesis of GTP

Thioester is cleaved (G~ -36 kJ/mol) drivingsubstrate level phosphorylation before Ox. Phos.

Recall: GTP + ADPGDP + ATP G~ 0 kJ/mol Effectively produces 1 ATP


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6. Succinate dehydrogenase: succinate oxidized (redox)

Tightly bound to inner mitochondrial membrane and ispart of complex II of the electron transport chain

FeS clusters provide a direct pathway for electronsto the ET chain leading to approximately 1.5 ATP

Malonate, a succinate analog, is a strong competitive inhibitor


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7. Fumarase: (fumarate hydratase) - hydration

Stereospecific to produce only trans L-malate The transition state is a carbanion


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8. L-Malate dehydrogenase: oxidation of malate (redox)

Oxaloacetate is quickly taken up in the cycle by citratesynthase

[Oxaloacetate]~10-6M makes the reaction favourable

(regenerated)


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Professor of Biochemistry at the University of Sheffield, 1945-1954,won the Nobel prize for Medicine/Physiology in 1953. Hediscovered the mechanism by which energy is released in livingcells through the oxidation of food stuffs, a cycle that becameknown as Krebs Cycle, citric acid cycle (CAC) or tricarboxylic acid

cycle (TCA).

SIR HANS KREBS

SIR HANS KREBSand

OTTO WARBURG

Lindau, 1966Nobel Prize Awards

Wh d h b i i t ?


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GlycolysisPhase 1

*

*

*

*

Where does each carbon originate?

Metabolism 221

Glucose C2 C5


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Metabolism 320

Glucose C2, C5*Glucose C1, C6

Now we canfollow thelabelled carbonsthrough the citricacid cycle

Experimentally,we would do thiswith radiolabels.

*

Figure 16-7

Glucose C2 C5 first round


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Metabolism 320

Glucose C2, C5

Lets follow the

carbon labelledblue through thecycle

- first round

Gl C2 C5 first round


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Metabolism 320

Glucose C2, C5

Lets follow the

carbon labelled

blue through thecycle

In the secondround of the

CAC, it islabelled as

- first round

- second round

* Glucose C1 C6 * first round


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Metabolism 320

What is the netenergy produced

by the Cycle?

Glucose C1, C6

*

Follow thecarbon labelledred through the

cycle What happens insubsequentrounds?

*

*

*

*

*

*

*

**

*

*

* - first round

P d t f t f CAC F 1 Gl


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Metabolism 320

Products of one turn of CAC For 1 Glucose:

1 NADH2.5 ATP(2x4x2.520 ATP)

1 FADH2

1.5 ATP(2x1x1.53 ATP)

1 GTP1 ATP(2x1x12 ATP)

_________25 ATP

Figure 16-13

NADH 2 e- go toETC I, III, IV10 H+pumped~ 2.5 ATP made

FADH22 e- go toETC II, III, IV6 H+pumped

~ 1.5 ATP made


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30.5 x 32 ATP = 978 kJ/mol for one glucose molecule% energy yielded from glucose = 976 kJ/mol/2840 kJ/mol ~ 34% (using G)

If we use GP, then we calculate a value of ~ 59% (see box 13-1 for GP)

4D The Amphibolic Nature of CAC (CAC as HUB of the cell)


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4D. The Amphibolic Nature of CAC (CAC as HUB of the cell)

The CAC intermediatesusually remain constantas a result of theseanaplerotic reactions

(shown as red arrows).

C


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Most Common Anaplerotic Reactions

Pyruvate carboxylase is regulated by acetyl CoA.If acetyl CoA is in excess, it activates the enzymeOtherwise the enzyme remains inactive.How Does this Enzyme Work?

**

P t


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Pyruvate

Carboxylase (PC)

Mechanism with

Biotin

Serves as a vitamin (deficiency dermatitis, glossitis)Prosthetic group to PC-LysNH2

Specialized carrier of CO2The carboxyl group is activated

by cleaving ATP and affixingCO2 to Biotin

Different active sites

for the two stepsBiotin swingsbetween the sites

4E Regulation of


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4E. Regulation ofthe CAC

These reaction are beingregulated by:

1. Substrate availability

2. Product Inhibition andAllosteric FeedbackInhibition

3. Covalent Modification

*

*

*

*

*

4E Regulation of the CAC


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1. Substrate availability - substrate availability varies with cellmetabolic state i.e. Citrate Synthase

2. Product Inhibition (P) and Allosteric Feedback Inhibition

a)All Dehydrogenases

- [NADH]/[NAD+] can be inhibited by mass action andby NADH competing with NAD+for binding - P

b) Citrate Synthase & -Ketoglutarate DH- Product inhibition by citrate and succinyl-CoA,

respectively - P

c) Citrate Synthase, Isocitrate & -Ketoglutarate DH- Allosteric feedback inhibition by NADH and/or ATP

4E. Regulation of the CAC

d) Pyruvate Dehydrogenase- Acetyl CoA competes with CoA for binding to E

2- P


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3. Covalent Modification through Ca2+signals

Pyruvate DH, Isocitrate DH, -Ketoglutarate DH

- Regulated by calcium (contraction signal) in muscleCa2+is stored in the SR, and its release is induced

by neurons. These enzymes are activated by Ca2+Example:Phosphorylation of PDH E1by pyruvate DH kinases (PDKs)

inactivates the enzymei) a) PDKs are activatedby ATP (signaling excess energy) OR

b) during starvation: glucose is required by the brain, socatabolism is blocked in muscle mitochondria by PDK activity that

phosphorylates and shuts down PDH

ii) In muscle, Ca2+-sensitive phosphatases can remove Pi

Increased cytosolic Ca2+Ca2+uptake by mitochondria

dephosphorylation activates Pyruvate Dehydrogenase.

Thus metabolism may be stimulated during exercise.

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