9 TCA CYCLE.ppt

Tricarboxylic Acid CycleTricarboxylic Acid Cycle

Dr Tolunimi Adedeji (MD)Dr Tolunimi Adedeji (MD)

I. OverviewI. Overview

The The tricarboxylic acid cycletricarboxylic acid cycle or or TCA cycleTCA cycle, also known as the , also known as the Krebs Krebs cyclecycle or or citric acid cyclecitric acid cycle, is an important component in metabolism, is an important component in metabolism

It is the converging point where oxidative metabolism of It is the converging point where oxidative metabolism of carbohydrates, amino acids, and fatty acids occurscarbohydrates, amino acids, and fatty acids occurs

These compounds are converted to COThese compounds are converted to CO22 and H and H22OO This oxidation facilitates the production of ATP, thus, an energy This oxidation facilitates the production of ATP, thus, an energy

source in most animalssource in most animals TCA cycle occurs exclusively in the mitochondria and is in close TCA cycle occurs exclusively in the mitochondria and is in close

proximity to reactions of electron transportproximity to reactions of electron transport The cycle is aerobic in nature (OThe cycle is aerobic in nature (O22 required as final electron acceptor) required as final electron acceptor) TCA cycle also involved in the formation of glucose from some amino TCA cycle also involved in the formation of glucose from some amino

acids and in formation of building blocks for synthesis of some amino acids and in formation of building blocks for synthesis of some amino acids and hemeacids and heme

Intermediates of the cycle can also be produced via catabolism of Intermediates of the cycle can also be produced via catabolism of some amino acidssome amino acids

II. Reactions of the TCA cycleII. Reactions of the TCA cycle

Oxaloacetate first undergoes Oxaloacetate first undergoes condensation upon reaction with condensation upon reaction with an acetyl group of acetyl CoAan acetyl group of acetyl CoA

Oxaloacetate is produced once Oxaloacetate is produced once more upon completion of the more upon completion of the cyclecycle


A.A. Oxidative decarboxylation Oxidative decarboxylation of pyruvateof pyruvate

Pyruvate (product of Pyruvate (product of aerobic glycolysis) is taken aerobic glycolysis) is taken to the mitochondrion prior to the mitochondrion prior to decarboxylation by a to decarboxylation by a pyruvate transporterpyruvate transporter

PyruvatePyruvate then converted to then converted to acetyl CoAacetyl CoA by by pyruvate pyruvate dehydrogenase complexdehydrogenase complex (multienzyme complex) – (multienzyme complex) – see figuresee figure


The pyruvate dehydrogenase complex consists of three enzymes:The pyruvate dehydrogenase complex consists of three enzymes:

1. pyruvate dehydrogenase (E1. pyruvate dehydrogenase (E11))

2. dihydrolipoyl transacetylase (E2. dihydrolipoyl transacetylase (E22))

3. dihydrolipoyl dehydrogenase (E3. dihydrolipoyl dehydrogenase (E33))

The complex also contains five coenzymes that serve as carriers The complex also contains five coenzymes that serve as carriers or oxidants for the intermediates of acetyl CoA (see figure on or oxidants for the intermediates of acetyl CoA (see figure on next slide); coenzymes are:next slide); coenzymes are:

1. thiamine pyrophosphate1. thiamine pyrophosphate2. lipoic acid2. lipoic acid3. coenzyme A3. coenzyme A4. FAD4. FAD5. NAD5. NAD++


Mechanism of action of the pyruvate dehydrogenase complex

TPP = thiamine pyrophosphate; L = lipoic acid


B.B. Synthesis of citrate from acetyl Synthesis of citrate from acetyl CoA and oxaloacetateCoA and oxaloacetate

This aldol condensation reaction This aldol condensation reaction requires the enzyme requires the enzyme citrate citrate synthasesynthase (see figure) (see figure)

The enzyme is allosterically The enzyme is allosterically activated by activated by CaCa2+2+ and ADP and ADP, and , and inhibited by inhibited by ATP, NADH, ATP, NADH, succinyl CoA, and fatty acyl CoA succinyl CoA, and fatty acyl CoA derivativesderivatives

Citrate inhibits Citrate inhibits phosphofructokinasephosphofructokinase, the , the rate-determining enzyme of rate-determining enzyme of glycolysis and activates glycolysis and activates acetyl acetyl CoA carboxylaseCoA carboxylase (rate-limiting (rate-limiting enzyme of fatty acid synthesis)enzyme of fatty acid synthesis)


C.C. Isomerization of citrateIsomerization of citrate

The enzyme The enzyme aconitaseaconitase facilitates the isomerization of facilitates the isomerization of citrate to isocitrate (see previous figure)citrate to isocitrate (see previous figure)

The enzyme is inhibited by The enzyme is inhibited by fluoroacetatefluoroacetate – a component – a component of rat poisonof rat poison

Fluoroacetate is converted to fluoroacetyl CoA, which Fluoroacetate is converted to fluoroacetyl CoA, which reacts with oxaloacetate to produce fluorocitrate; citrate reacts with oxaloacetate to produce fluorocitrate; citrate levels then riselevels then rise


D.D. Oxidation and decarboxylation of isocitrateOxidation and decarboxylation of isocitrate

Oxidative decarboxylation of isocitrate is carried out in Oxidative decarboxylation of isocitrate is carried out in the presence of the presence of isocitrate dehydrogenaseisocitrate dehydrogenase to give to give --ketoglutarateketoglutarate, as well as one of the three NADH , as well as one of the three NADH molecules produced by the cycle, and a COmolecules produced by the cycle, and a CO22 molecule molecule (see figure)(see figure)

This reaction is one of the rate-determining steps of the This reaction is one of the rate-determining steps of the cyclecycle

Enzyme is allosterically activated by Enzyme is allosterically activated by ADP and CaADP and Ca2+2+, and , and is inhibited by is inhibited by ATP and NADHATP and NADH


E.E. Oxidative decarboxylation of Oxidative decarboxylation of --ketoglutarateketoglutarate

This reaction is achieved in the This reaction is achieved in the presence of presence of -ketoglutarate -ketoglutarate dehydrogenase complexdehydrogenase complex to produce to produce succinyl CoAsuccinyl CoA – see figure – see figure

This reaction releases the second COThis reaction releases the second CO22 and NADH molecules of the cycleand NADH molecules of the cycle

Coenzymes required are Coenzymes required are thiamine thiamine pyrophosphatepyrophosphate, , lipoic acidlipoic acid, , FADFAD, , NADNAD++, and , and coenzyme Acoenzyme A

The enzyme is inhibited by The enzyme is inhibited by ATP, GTP, ATP, GTP, NADH, and succinyl CoA NADH, and succinyl CoA and and activated by activated by CaCa2+2+


F.F. Cleavage of succinyl CoACleavage of succinyl CoA

The enzyme The enzyme succinate thiokinasesuccinate thiokinase is responsible for the is responsible for the cleavage of cleavage of succinyl CoAsuccinyl CoA to give to give succinatesuccinate and and CoACoA, see , see previous figureprevious figure

This reaction is coupled to phosphorylation of GDP to GTPThis reaction is coupled to phosphorylation of GDP to GTP

G.G. Oxidation of succinateOxidation of succinate

SuccinateSuccinate is oxidized to is oxidized to fumaratefumarate by by succinate succinate dehydrogenasedehydrogenase to give the reduced coenzyme FADH to give the reduced coenzyme FADH22 from FAD (see previous figure)from FAD (see previous figure)

The enzyme is inhibited by oxaloacetateThe enzyme is inhibited by oxaloacetate


H.H. Hydration of fumarateHydration of fumarate

FumarateFumarate is hydrated to is hydrated to malatemalate by by fumarasefumarase (see previous (see previous figure)figure)

Fumarate is also produced by the Fumarate is also produced by the urea cycle, in purine synthesis, urea cycle, in purine synthesis, and during catabolism of amino and during catabolism of amino acids (phenylalanine and tyrosine)acids (phenylalanine and tyrosine)

I.I. Oxidation of malateOxidation of malate

MalateMalate is oxidized to is oxidized to oxaloacetateoxaloacetate in the presence of in the presence of malate dehydrogenasemalate dehydrogenase (see (see figure)figure)

Reaction affords the third and Reaction affords the third and final NADH of the cyclefinal NADH of the cycle

III. Energy produced by the TCA III. Energy produced by the TCA cyclecycle

Total yield of ATP from the oxidation of one acetyl CoA Total yield of ATP from the oxidation of one acetyl CoA shown in figure 9.8 belowshown in figure 9.8 below

Three pairs of electrons transferred reducing NADThree pairs of electrons transferred reducing NAD++ to to NADH and one pair reducing FAD to FADHNADH and one pair reducing FAD to FADH22

IV. Regulation of the TCA cycleIV. Regulation of the TCA cycle

regulation can occur by activation regulation can occur by activation and inhibition of enzyme activities and inhibition of enzyme activities (see figure)(see figure)

This is different to glycolysis, since This is different to glycolysis, since phosphofructokinase primarily phosphofructokinase primarily regulates glycolysisregulates glycolysis

The regulated enzymes include The regulated enzymes include citrate synthasecitrate synthase, , isocitrate isocitrate dehydrogenasedehydrogenase, and , and --ketoglutarate dehydrogenase ketoglutarate dehydrogenase complexcomplex

Regulation may also arise by the Regulation may also arise by the availability of ADP – elevated ADP availability of ADP – elevated ADP activates TCA cycle; low levels of activates TCA cycle; low levels of ADP lowers activity of TCA cycleADP lowers activity of TCA cycle

Remember that regulation of the TCA cycle. Remember that regulation of the TCA cycle. like that of glycolysis, occurs at both the level like that of glycolysis, occurs at both the level of entry of substrates into the cycle as well as of entry of substrates into the cycle as well as at the key reactions of the cycle. at the key reactions of the cycle.

Fuel enters the TCA cycle primarily as acetyl-Fuel enters the TCA cycle primarily as acetyl-CoA. CoA.

The generation of acetyl-CoA from The generation of acetyl-CoA from carbohydrates is, therefore, a major control carbohydrates is, therefore, a major control point of the cycle. point of the cycle.

This is the reaction catalyzed by the PDH This is the reaction catalyzed by the PDH complex. complex.

By way of review, the PDH complex is inhibited by acetyl-By way of review, the PDH complex is inhibited by acetyl-CoA and NADH and activated by non-acetylated CoA CoA and NADH and activated by non-acetylated CoA (CoASH) and NAD(CoASH) and NAD++. .

The pyruvate dehydrogenase activities of the PDH complex The pyruvate dehydrogenase activities of the PDH complex are regulated by their state of phosphorylation. are regulated by their state of phosphorylation.

This modification is carried out by a specific kinase (PDH This modification is carried out by a specific kinase (PDH kinase) and the phosphates are removed by a specific kinase) and the phosphates are removed by a specific phosphatase (PDH phosphatase). phosphatase (PDH phosphatase).

The phosphorylation of PDH inhibits its activity and, The phosphorylation of PDH inhibits its activity and, therefore, leads to decreased oxidation of pyruvate. PDH therefore, leads to decreased oxidation of pyruvate. PDH kinase is activated by NADH and acetyl-CoA and inhibited kinase is activated by NADH and acetyl-CoA and inhibited by pyruvate, ADP, CoASH, Caby pyruvate, ADP, CoASH, Ca2+2+ and Mg and Mg2+2+. .

The PDH phosphatase, in contrast, is activated by MgThe PDH phosphatase, in contrast, is activated by Mg2+2+ and Caand Ca2+2+..

Since three reactions of the TCA cycle as well as PDH Since three reactions of the TCA cycle as well as PDH utilize NADutilize NAD++ as co-factor it is not difficult to understand as co-factor it is not difficult to understand why the cellular ratio of NADwhy the cellular ratio of NAD++/NADH has a major impact /NADH has a major impact on the flux of carbon through the TCA cycle.on the flux of carbon through the TCA cycle.

Substrate availability can also regulate TCA flux. Substrate availability can also regulate TCA flux. This occurs at the citrate synthase reaction as a result of This occurs at the citrate synthase reaction as a result of

reduced availability of oxaloacetate. reduced availability of oxaloacetate. Product inhibition also controls the TCA flux, e.g. citrate Product inhibition also controls the TCA flux, e.g. citrate

inhibits citrate synthase, α-KGDH is inhibited by NADH inhibits citrate synthase, α-KGDH is inhibited by NADH and succinyl-CoA. and succinyl-CoA.

The key enzymes of the TCA cycle are also regulated The key enzymes of the TCA cycle are also regulated allosterically by Caallosterically by Ca2+2+, ATP and ADP. , ATP and ADP.

Key concept map for tricarboxylic Key concept map for tricarboxylic acid cycleacid cycle

Any Questions?Any Questions?

9 TCA CYCLE.ppt

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