krebs cycle

1

Amity Institute of Microbial Technology

Kreb’s Cycle/Citric Acid

Cycle/TCA Cycle

By

Dr Shwet Kamal

2

Amity Institute of Microbial Technology

3

Amity Institute of Microbial Technology

CITRIC ACID CYCLE = TCA CYCLE = KREBS CITRIC ACID CYCLE = TCA CYCLE = KREBS CYCLECYCLE Definition:

-- Acetate in the form of acetyl-CoA, is derived from pyruvate and other metabolites, and is oxidized to CO2

in the citric acid cycle.

4

Amity Institute of Microbial Technology

One high energy compound is produced for each cycle.

The electrons from the TCA cycle are made available to an electron transport chain in the form of three NADH and one FADH2 and ultimately energy is provided foroxidative phosphorylation.

5

Amity Institute of Microbial Technology

The citric acid cycle is central to all respiratory oxidation, oxidizing acetyl-CoA from glucose, lipid and protein catabolism in aerobic respiration to maximize energy gain.The cycle also supplies some precursors for biosynthesis.All enzymes are in the mitochondrial matrix or inner mitochondrial membrane

6

Amity Institute of Microbial Technology

The three stagesthree stages of cellular respiration:Stage 1. Acetyl CoA production from glucose, fatty acids and amino acidsStage 2. Acetyl CoA oxidation =TCA Cycle = yielding reduced electron carriersStage 3. Electron transport and oxidative phosphorylation oxidation of these carriers and production of ATP

7

Amity Institute of Microbial Technology

Acetyl CoAAcetyl CoA

HS-CoA Acety

l

8

Amity Institute of Microbial Technology

Conversion of pyruvate to acetyl-CoA -- A major Stage 1 setup Enzyme = pyruvate dehydrogenase pyruvate dehydrogenase complexcomplexLocation = mitochondrial matrix

CH3 CH3

C=O + NAD++ HS-CoA C=O +NADH+CO2

COO- S-CoA 

Pyruvate Acetyl-CoA

9

Amity Institute of Microbial Technology

Irreversible  -- irreversible means acetyl-CoA cannot be converted backward to pyruvate;

hence “fat cannot be converted to carbohydrate”

10

Amity Institute of Microbial Technology

Complex of 2.5 x 106 Da, including multiple copies of three enzymes  pyruvate dehydrogenase (=E1) has coenzyme = thiamine pyrophosphatethiamine pyrophosphate (TPP) (TPP)

-- TPP is coenzyme for all decarboxylations of -keto acids. -- lack of thiamine = beriberi  dihydrolipoyl transacetylase (=E2) has coenzymes lipoatelipoate and CoA

11

Amity Institute of Microbial Technology

dihydrolipoyl dehydrogenase (= E3) has coenzymes FAD and NAD+

S--S--

TPP FAD 

E1 E2 E3 N A D+

12

Amity Institute of Microbial Technology

lipoic acid lipoic acid akaaka lipoamidelipoamide

thiamine pyrophosphate (TPP)thiamine pyrophosphate (TPP)

13

Amity Institute of Microbial Technology

Partial reactions of PDH: introduction of pyruvate onto TPP in E1

hydroxyethyl-TPP  H H O O CO2 O-O-C-C-CH3 H-CCH3

TPP E1

14

Amity Institute of Microbial Technology

Transfer to lipoamide of E2

hydroxyethyl-TPPhydroxyethyl-TPP + + O CH3 + TPP S C S S HS E2 E2

lipoamide-E2 acetyl-lipoamide-E2

(disulfide,oxidized) (reduced) Note concurrent oxidation to acetyl and reduction of S

15

Amity Institute of Microbial Technology

E2 then transfers acetyl to CoA; acetyl-

CoA leaves.  E3 uses its bound coenzyme FAD to

oxidize lipoamide back to disulfide and generating FADH2.  FAD is recovered from FADH2 via

reducing NAD to NADH. An NADHNADH is generated.

16

Amity Institute of Microbial Technology

Regulation of Pyruvate DehydrogenaseRegulation of Pyruvate DehydrogenaseIrreversible reaction must be tightly controlled-- three ways1. Allosteric Inhibition-- inhibited by products: acetyl-CoA and NADH -- inhibited by high ATP

2. Allosteric activation by AMP Ratio ATP/AMP important

17

Amity Institute of Microbial Technology

3. Phosphorylation/dephosphorylation of Phosphorylation/dephosphorylation of EE11 subunit subunit   Pi E1-OH ATP (active)phosphatase kinase activator = activator = insulin acetyl-CoAindirectly NADH, not

cAMP  H2O E1-OP ADP

(inactive)kinase inhibitors = pyruvate, ADP

18

Amity Institute of Microbial Technology

Formation of citrate

• Oxaloacetate condenses with acetyl CoA to form Citrate

• Non-equilibrium reaction catalysed by citrate synthase– Inhibited by:

• ATP• NADH• Citrate - competitive

inhibitor of oxaloacetate

19

Amity Institute of Microbial Technology

Citrate isocitrate• Citrate isomerised to

isocitrate in two reactions (dehydration and hydration)

• Equilibrium reactions catalysed by aconitase

• Results in interchange of H and OH– Changes structure and

energy distribution within molecule

• Makes easier for next enzyme to remove hydrogen

20

Amity Institute of Microbial Technology

isocitrate -ketoglutarate

• Isocitrate dehydrogenated and decarboxylated to give -ketoglutarate

• Non-equilibrium reactions catalysed by isocitrate dehydrogenase

• Results in formation of:– NADH + H+

– CO2

• Stimulated (cooperative) by isocitrate, NAD+, Mg2+, ADP, Ca2+ (links with contraction)

• Inhibited by NADH and ATP

21

Amity Institute of Microbial Technology

-ketoglutarate succinyl CoA

• Series of reactions result in decarboxylation, dehydrogenation and incorporation of CoASH

• Non-equilibrium reactions catalysed by -ketoglutarate dehydrogenase complex

• Results in formation of:

– CO2

– NADH + H+

– High energy bond

• Stimulated by Ca2+

• Inhibited by NADH, ATP, Succinyl CoA (prevents CoA being tied up in matrix)

22

Amity Institute of Microbial Technology

Succinyl CoA succinate

• Equilibrium reaction

catalysed by succinate

thiokinase

• Results in formation of:

– GTP

– CoA-SH

23

Amity Institute of Microbial Technology

Succinate fumarate

• Succinate

dehydrogenated to form

fumarate

• Equilibrium reaction

catalysed by succinate

dehydrogenase

– Only Krebs enzyme

contained within inner

mitochondrial

membrane

• Results in formation of

FADH2From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random

House p 550.

24

Amity Institute of Microbial Technology

Fumarate malate

• Fumarate hydrated to

form malate

• Equilibrium reaction

catalysed by fumarase

• Results in redistribution

of energy within

molecule so next step

can remove hydrogenFrom: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random

House p 550.

25

Amity Institute of Microbial Technology

Malate oxaloacetate

• Malate dehydrogenated

to form oxaloacetate

• Equilibrium reaction

catalysed by malate

dehydrogenase

• Results in formation of

NADH + H+

From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 550.

26

Amity Institute of Microbial Technology

Regulation of Krebs Cycle

• Cycle always proceeds

in same direction due to

presence of 3 non-

equilibrium reactions

catalysed by

– Citrate synthase

– Isocitrate

dehydrogenase

-ketoglutarate

dehydrogenase

From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 550.

27

Amity Institute of Microbial Technology

Regulation of Krebs Cycle• Flux through KC increases

during exercise

• 3 non-equilibrium enzymes inhibited by NADH

– KC tightly coupled to ETC• If NADH decreases due to

increased oxidation in ETC flux through KC increases

• Isocitrate dehydrogenase and -ketoglutarate dehydrogenase also stimulated by Ca2+

– Flux increases as contractile activity increases

From: Summerlin LR (1981) Chemistry for the Life Sciences. New York: Random House p 550.

28

Amity Institute of Microbial Technology

Overall ReactionOverall Reaction:

acetyl-CoA+3NAD++FAD+GDP+Pi+2H2O2CO2 + 3NADH + FADH2 +GTP+2H++CoA

one high energy compound made 

four pairs of electrons are madeavailable to the respiratory chain andoxidative phosphorylation. These areused to generate most of the ATPneeded.

29

Amity Institute of Microbial Technology

What is the What is the maximum yield maximum yield of high energy of high energy ATP in the aerobic catabolism of glucose?ATP in the aerobic catabolism of glucose?

GlycolysisGlycolysis::glucose 2pyruvate + 2NADH+2ATP 8 ATPs

Pyruvate Dehydrogenase:Pyruvate Dehydrogenase:2pyruvate 2acetyl CoA + 2NADH 6 ATPs

TCA cycle:TCA cycle:acetyl CoA2CO2+3NADH+FADH2+GTP 2x12ATPs

 OVERALL yield from glucose 38 ATPs38 ATPs

30

Amity Institute of Microbial Technology

ENERGY RELATIONSHIPS ENERGY RELATIONSHIPS G° for oxidation of glucose to CO2 is 2,840 kJ/mole  Much of this energy conserved as ATP 38 ATP X 30.5 kJ/mole ATP =1,160 kJ/mole glucose  This represents 41% 41% conservation of the potential energy available in glucose as ATP.===================================

31

Amity Institute of Microbial Technology Amphibolic pathwaysAmphibolic pathways

a- transaminasestransaminasesoxaloacetate Asp removes 4C-ketoglutarate Glu removes 5Cpyruvate Ala removes 6Cb- fatty acid biosynthesisfatty acid biosynthesiscitrate acetyl CoA and oxaloacetate acetyl CoA can build fatty acidsc- heme biosynthesisheme biosynthesissuccinyl CoA + glycine porphyrins

32

Amity Institute of Microbial Technology Anaplerotic reactionsAnaplerotic reactions a-a- pyruvate carboxylase - replacesoxaloacetate- most important,especially in liver and kidney. OCH3-C-COO- + CO2 + ATP O -OOC-CH2C-COO- + ADP + Pi

oxaloacetate Note: same rxn in gluconeogenesis

33

Amity Institute of Microbial Technologybb- malic enzyme - replaces malate-- pyruvate + CO2 + NADPHmalate + NADP+

cc- from amino acids reversals of transaminations -- restores oxaloacetate or-ketoglutarate with abundant asp or glu glutamate dehydrogenaseglu + NAD(P)+ -ketoglutarate + NAD(P)H + NH4

+

34

Amity Institute of Microbial TechnologyTHE PENTOSE PHOSPHATE SHUNTTHE PENTOSE PHOSPHATE SHUNTDefinition:Oxidation of glucose to provideNADPH and ribose 5-phosphate, bothrequired for biosynthesis. (hexosephosphate shunt)Note: Electron carrier NAD+/NADHused primarily to oxidize substrates. Electron carrier NADP+/NADPHused to reduce substrates & functionssynthetically, e.g., steroids, cholesteroland fatty acid synthesis.

35

Amity Institute of Microbial Technology

Functions:1- generates reducing power in cytosol (NADPH) for fat & steroid biosynthesis 2- generates pentoses and pentosephosphates3- provides catabolic path for dietarypentose metabolism and for generationof aldose and ketose families ofC3, C4, C5, and C7 carbohydrates.4- CO2 fixation (dark reactions ofphotosynthesis) in plants.

36

Amity Institute of Microbial Technology

Tissue location in animals:  high abundance in three organs= liver, lactating mammary gland andadrenals not much in muscle cells- notaccumulate fatty acids in muscle cells

Within cells in cytosol & enzymes are soluble

Two phases of pentose phosphate shunt

37

Amity Institute of Microbial Technology

Oxidative PhaseOxidative Phase-generates reducing power as NADPH  glucose-6-phosphateNADP+ glucose-6-phosphate NADPH+H+ dehydrogenase C=O 6-phosphoglucono HC-OH --lactone HO-CH O unstable with a HC-OH large release of HC energy upon

CH2O P hydrolysis

38

Amity Institute of Microbial Technology C=O 6-phosphoglucono

H COH --lactone HOCH O HCOH HC CH2O P H2O lactonase COO- sugar acid- CHOH -pulls initial HOCH dehydrogenation CHOH CHOH6-phosphogluconateCH2O P

39

Amity Institute of Microbial Technology 6-phosphogluconateNADP+ 6-phosphogluconate NADPH+H+ dehydrogenase oxidative CO2 decarboxylation CH2OH from C-1 C=O HCOH D-ribulose-5-phosphate HCOH 5C-ketose CH2OP

40

Amity Institute of Microbial Technology

Isomerization H H H H H H O O O O H O O O O HHC C C C COP C C C C COP H H H H H H H H H H D-ribulose D-ribose phosphate phosphate 5C ketose 5C aldose

Enzyme = ribose phosphate isomerase-- Only a small amount of the ribulosephosphate is isomerized-- endiol intermediate as G6P to F6P

41

Amity Institute of Microbial Technology Summary of First PhaseSummary of First PhaseOverall reaction:glucose-6- P + 2 NADP+ + H2O ribose-5- P +CO2 + 2 NADPH + 2H+

 -- this reductive portion of the shunthas generated reducing power (NADPH) and 5-carbon sugars-- Most cells need much moreNADPH than pentoses

42

Amity Institute of Microbial Technology Nonoxidative PhaseNonoxidative Phase-- three pentose phosphate moleculesare transformed into two hexosephosphates and a triose phosphate. -- uses only three enzymes =ribulose-(P) 3-epimerase,transketolase, transaldolase.