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 Sheet 5, Dr.Nafeth, by Faten Jbara 14\2\2012

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Majed Sharayha



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PostSubject: Sheet 5, Dr.Nafeth, by Faten Jbara 1422012   Thu Feb 16, 2012 4:46 am

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BY THE NAME OF GOD

Tricarboxylic Acid Cycle

** A series of enzymatic reactions involving oxidative metabolism of acetyl units and producing high-energy phosphate compounds, which serve as the main source of cellular energy. Also called citric acid cycle, Krebs acid cycle.

** accounts for two thirds of the ATP generated in the body by producing NADH&FAD(2H)

** all pathways for oxidation of fatty acids,carbohydrates,amino acids & ketone bodies generate acetyl CoA

** acetyl CoA is considered as the first substrate of this cycle

** why it is called tricarboxylic acid cycle??!
this name denotes the involvement of the tricarboxylates citrate & isocitrate

** also called>> • Krebs cycle: because of the German scientist Hans Krebs who first formulated it's reactions into a cycle

• citric acid cycle: because citrate was one of the first compounds known to participate

** starts with the combination between acetyl group & oxaloacetate

** oxaloacetate(4 carbons) + acetyl CoA → citrate ( 6 carbons)
** citrate will be converted into isocitrate

** generally TCA cycle gives>>
electrons carried by 3NADH & 1 FAD(2H) to make ATP in the oxidative phosphorylation + 1 molecule of GTP+2 CO2 molecules (getting out in 2 steps)


** the rate of TCA cycle is regulated by the products of the cycle itself >> by NADH/NAD+ ratio, FAD(2H)/FAD ratio, Ca+2 concentration & ATP(can control all energy-requiring pathways in the body)

** What are the consequences when there is a defect in the Krebs cycle??!
--- inability to generate ATP from fuel oxidation because there will not be NADH or FAD(2H)
--- accumulation of the TCA cycle precursors (intermediates)& this depend on the site of this defect in the cycle >> for example: any defect in the transition from α-ketoglutarate into succinyl-CoA then there is accumulation of α-ketoglutarate & so on.

** making high- energy molecules like ATP,GTP,CTP without using O2(without going through oxidative phosphorylation) is called >> substrate level phosphorylation
** making them by using O2 >> oxidative phosphorylation


REACTIONS OF THE TCA CYCLE

♦ STEP 1 >>

Enzyme : citrate synthase
Reaction : acetyl portion of acetyl CoA combines with the 4- carbon molecule oxaloacetate
Products : citrate ( 6-carbon molecule)

♦STEP 2 >>
Enzyme: aconitase
Reaction: the hydroxyl(alcohol) group of citrate is moved to an adjacent carbon so that it can be oxidized to form a keto group
Products: isocitrate

♦STEP 3 >>
Enzyme: isocitrate dehydrogenase
Reaction: oxidation of alcohol group in isocitrate into carbonyl group to form keto acid , this reaction is called decarboxylation reaction(carboxyl group is removed from isocitrate out of the cycle as CO2)
Products : the first CO2 molecule + the first NADH molecule+ α-ketoglutarate (keto acid because it has ketone group & carboxyl group)

♦STEP 4 >> α-ketoglutarate to succinyl CoA

Enzyme: α-ketoglutarate dehydrogenase complex
Reaction: oxidative decarboxylation of α-ketoglutarate to succinyl-CoA in which one of the carboxyl groups of α- ketoglutarate is released as CO2(decarboxylation) ,then one hydrogen is removed(dehydrogenase) & this hydrogen is carried on NAD+ to become NADH
- keto group in α-ketogultarate is oxidized to an acidic group which combines with CoASH to form succinyl-CoA (has high-energy thioester bond )
Products: the second CO2 molecule+ the second NADH molecule+ succinyl CoA

♦ STEP 5 >> succinyl CoA to succinate

Enzyme: succinate thiokinase (removes thiol group)
Reaction: this enzyme removes SH (thiol group) from succinyl- CoA producing GTP(as mentioned above)
Products: 1GTP molecule+CoASH+ succinate


generation of GTP >>٭٭
thioester bond in succinyl CoA is high-energy bond → it's not stable→degredation to become stable→release energy→this energy is used to combine GDP with Pi to form GTP (this reaction is an example on substrate level phosphorylation)

notes: - high-energy bonds(P-P/ P-C / S-C) ***
- although the processes of TCA cycle are not involving any O2 it gives 2 CO2 molecules

♦STEP 6 >> succinate to fumarate

Enzyme: succinate dehydrogenase
Reaction: this enzyme removes 2 hydrogens from the middle carbon in succinate forming fumarate with double bond
Products: 1 FAD(2H) molecule + fumarate





♦ STEP 7 >> fumarate to malate

Enzyme:fumarase
Reaction: hydrolysis reaction of fumarate by this enzyme through addition of water molecule (H2O)
- so the 2 carbons of the double bond one of them will take OH̄ group & the other will take proton from this water molecule forming malate
Products: malate



♦ STEP 8 >> malate to oxaloacetate

Enzyme: malate dehydrogenase
Reaction: alcohol group of malate is oxidized to keto group through donation of electrons to NAD+ forming oxaloacetate again
Products: the third NADH molecule


** with regeneration of oxaloacetate the TCA cycle is completed 

** because oxaloacetate is regenerated with each turn of the cycle ,it's not considered as substrate of the TCA cycle( it is getting used & getting regenerated again ).

** the change occurs on oxaloacetate that it's not the same carbons which enter the reaction getting out of the reaction.



COENZMES of the cycle

** the enzymes of the TCA cycle depend on coenzymes which are helper molecules that enzyme need to be active.

** Coenzymes can be organic, metallic or organometalic for example: quinones & heme in hemoglobin (heme is organometallic coenzyme)

** α-ketoglutarate dehydrogenase complex(which converts α-ketoglutarate into succinyl CoA) uses thiamine pyrophosphate,lipoate& FAD as coenzyme


Coenzymes in this cycle>>
A) 1- FAD
2- NAD+

NAD+ FAD
Accepts pair of electrons from the same source Accepts single electrons from 2 different sources
Doesn't generate free radicals It forms free radicals that can affect the cell & the mitochondria itself
Found in free pool (not bound to enzyme) Must remain very tightly attached to it's enzyme, to prevent the effect of the free radicals
E̊' is fixed E̊' for enzyme- bound FAD varies greatly
Plays regulatory role in the cycle more than FAD, because it is in free pool inside the cell so it can hit more than one pathway Doesn't play regulatory role because it remains tightly attached to it's enzyme so it cannot generate effects like NAD+
Produced by:
- the first one by isocitrate dehydrogenase
- second one by α-ketoglutarate dehydrogenase
-third one by malate dehydrogenase Produced by succinate dehydrogenase enzyme


E̊' for NAD+ is fixed > because it's found in free pool in the cell so we can measure its voltage

E̊' for FAD varies greatly > because voltage arises from chemical bonds → chemical bonds contain electrons →FAD present in different enzymes →so chemical bonds between FAD & enzyme itself are different from enzyme to enzyme →different voltage E̊'

> what are the factors that affect the voltage of FAD?
1- the way in which FAD binds the protein scaffolds
2- the amino acids that FAD binds
3- whether around it > β sheet or α
4- whether the polypeptide is free in movement or fixed


B) CoASH

**Acylation coenzyme participates in the reactions through the formation of thioester bond between sulfur of CoASH & an acyl group (acetyl CoA,succinyl-CoA)

**thioester bond differs from oxygen ester bond ,because sulfur(S) doesn't share it's electrons as the oxygen => so that thioester bond is high-energy bond that has a large negative ΔG̊' of hydrolysis (-13kcal/mole) => this energy is used to make GTP

** the more the excess energy >> the cycle moves in forward direction


C) Coenzymes in α-ketoacid dehydrogenase complexes

** Three-member family of similar α-ketoacid dehydrogenase complexes >> 1- α-ketoglutarate dehydrogenase complex
2- pyruvate dehydrogenase complex
3- α-ketoacid dehydrogenase complex

** These 3 complexes work in the same way & have the same structures so that they are in one group

** all of the α-ketoacid dehydrogenase complexes are huge enzyme complexes composed of multiple subunits of 3 different enzymes (E1,E2&E3)>>the product goes from E1 to E2 to E3




E1:
* α-ketoacid decarboxylase which contains coenzyme thiamine pyrophosphate (TPP)
* removes the carboxyl group of the α-ketoglutarate producing CO2
* Thiamine is an external material from food
* TPP in α-ketoacid dehydrogenase combines the acyl group which found in α-ketoglutarate removing carboxyl group out as CO2 "circle 1"




E2:
* transacylase containing coenzyme lipoate (lipoate has 2 sulfur atoms forming disulfide bond)
* lipoate binds the acyl group from thiamine in α-ketoacid dehydrogenase →TPP returns as it was & acyl group binds one sulfur atom & the other sulfur atom binds H atom (disulfide bond is broken )
"circle 2 in the picture above"

- E2 is transacylase so now it transfers the acyl group from lipoate to CoASH→CoASH binds the acyl group from lipoate →lipoate is reduced






E3:
* dihydrolipoyl dehydrogenase contains coenzyme FAD
* it transfers 2 hydrogens from reduced lipoate so >>
- disulfide bond is reformed in lipoate
- E3 takes these 2 hydrogens to it's tightly bound FAD →FAD(2H) →FAD(2H) gives the 2 electrons (hydrogens) to NAD+ to form NADH "circle 3 above "



NOTES:

** so when there are 3 enzymes bound together at the same place ,this will accelerate the reaction & prevent the loss of energy .

** TPP binds magnesium weakly so it can dissociate easily from the enzyme & can bind easily too =>this mean that it has high turnover rate=> so any deficiency of thiamine in food => TCA cycle will be affected=> no TPP=>no decarboxylation reaction=>substrate (α-ketoglutarate) of the reaction will be more=>α-ketoglutarate,pyruvate & other α-ketoacids accumulate in the blood.

** lipoate is synthesized from carbohydrates & amino acids ,amino acids provide the nitrogen & other carbon bonds are from carbohydrates


ENERGITICS OF THE TCA CYCLE

** Total amount of energy available from acetyl group is (228kcal/mole)
** Energy from high- energy bonds that formed :
3 NADH > 3*53=159kcal/mole
1 FAD(2H) = 41 kcal/mole
1 GTP = 7 kcal/mole

The sum= 159+41+7=207 kcal/mole

So the efficiency of the TCA cycle >>
207/228*100%= 90%

TCA cycle is extremely efficient.


WHAT MAKES THE REACTION MOVING FORWARD ??!

** There are 3 reactions in this cycle with 3 large negative ΔG:
1- from oxaloacetate to citrate
2- from isocitrate to α-ketoglutarate
3- from α-ketoglutarate to succinyl CoA

=> 3 large negative ΔG so that the reaction goes in forward direction

The reactions of the TCA cycle are not reversible >> because the products are frequently used ( concentration of the products is low → more negative ΔG→ irreversible reactions )

00000000000000000000000000000000000000000000000000000000


BIOCHEMISTRY lec # 5
DATE: 14/2/2012





•When life gets harder, know that you've just leveled up ^_^

•DONE BY : FATEN JBARA
•Special thanks to Rand Al Kilani & Rana Khlefat

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