CITRIC ACID CYCLE- discovered byCITRIC ACID CYCLE- discovered by Sir Hans KrebsSir Hans Krebs in 1953. He was awarded in 1953. He was awarded

Nobel Prize in MedicineNobel Prize in Medicine

1.1. The The citric acid cyclecitric acid cycle (also known as the (also known as the tricarboxylic acid cycletricarboxylic acid cycle, the , the TCATCA cycle, cycle, or the or the Krebs cycleKrebs cycle) is a series of ) is a series of chemical reactionschemical reactions of central importance in of central importance in all living all living cellscells that utilize that utilize oxygenoxygen as part of as part of cellular respirationcellular respiration. .

2.2. In In aerobic organismsaerobic organisms, the citric acid cycle is , the citric acid cycle is part of a part of a metabolic pathwaymetabolic pathway involved in the involved in the chemical conversion of chemical conversion of carbohydratescarbohydrates, , fatsfats and and proteinsproteins into into carbon dioxidecarbon dioxide and and waterwater to generate a form of usable energy. to generate a form of usable energy.

3.3. It is the second of three metabolic It is the second of three metabolic pathways that are involved in pathways that are involved in fuel moleculefuel molecule catabolismcatabolism and and ATPATP production, the other production, the other two being two being glycolysisglycolysis and and oxidative phosphorylationoxidative phosphorylation..

4.4. The citric acid cycle also provides The citric acid cycle also provides precursors for many compounds such as precursors for many compounds such as certain certain amino acidsamino acids, and some of its , and some of its reactions are therefore important even in reactions are therefore important even in cells performing cells performing fermentationfermentation..

ACETYL-CoA (Acetyl Coenzyme A, ACETYL-CoA (Acetyl Coenzyme A, ACoA)ACoA) Catabolism of carbohydrates, fatty acids, Catabolism of carbohydrates, fatty acids,

amino acids releases energy from ACoA amino acids releases energy from ACoA During glycolysis, glucose is converted to During glycolysis, glucose is converted to

pyruvatepyruvate which is converted to ACoA by a which is converted to ACoA by a group of enzymes known as group of enzymes known as pyruvate pyruvate dehydrogenase complexdehydrogenase complex

NADH is produced during those processNADH is produced during those process In TCA two carbon atoms of the acetyl-In TCA two carbon atoms of the acetyl-

Coenzyme A are ultimately oxidized to CO2Coenzyme A are ultimately oxidized to CO2 ACoA is a carrier of ACoA is a carrier of acyl acyl groups and the groups and the

catalyzed steps of TCA occurs in the catalyzed steps of TCA occurs in the mitochondria.mitochondria.

CITRIC ACID CYCLECITRIC ACID CYCLE

REACTIONS OF THE CITRIC ACID REACTIONS OF THE CITRIC ACID CYCLECYCLE

1.1. CITRIC SYNTHASE catalyses the CITRIC SYNTHASE catalyses the condensationcondensation of ACoA with oxaloacetate to form of ACoA with oxaloacetate to form citratecitrate. . The reaction is reversible, but is a main The reaction is reversible, but is a main regulatory point. A low NAD+/NADH ratio and regulatory point. A low NAD+/NADH ratio and succinyl- CoA succinyl- CoA inhibitsinhibits its activity its activity

2.2. ACONITASE reversibly catalyses the ACONITASE reversibly catalyses the conversion of citrate to conversion of citrate to isocitrateisocitrate

3.3. ISOCITRATE DEHYDROGENASE oxidatively ISOCITRATE DEHYDROGENASE oxidatively decarboxylates isocitrate to decarboxylates isocitrate to αα-ketoglutarate-ketoglutarate (2-oxyglutarate). In this process NAD+ s (2-oxyglutarate). In this process NAD+ s reduced to NADH and CO2 is released. reduced to NADH and CO2 is released. Isocitrate dehydrogenase is Isocitrate dehydrogenase is inhibitedinhibited by ATP by ATP and NADH and and NADH and activatedactivated by ADP and NAD+ by ADP and NAD+

REACTIONS contd.REACTIONS contd.4.4. αα-KETOGLUTARATE DEHYDROGENASE produces -KETOGLUTARATE DEHYDROGENASE produces

succinyl- CoAsuccinyl- CoA from from αα-ketogluturate and -ketogluturate and coenzyme A. Another NAD+ is reduced to NADH coenzyme A. Another NAD+ is reduced to NADH and CO2 is released. Both NADH and succinyl-and CO2 is released. Both NADH and succinyl-CoA inhibit the enzyme complex, CoA inhibit the enzyme complex, 2-oxygluturate 2-oxygluturate dehyrogenase complexdehyrogenase complex

5.5. SUCCINYL-CoA SYNTHETASE converts succinyl-SUCCINYL-CoA SYNTHETASE converts succinyl-CoA to CoA to succinatesuccinate. GDP (glyceralaldehyde di-. GDP (glyceralaldehyde di-phosphate) is converted to GTP during this phosphate) is converted to GTP during this step. This is the only step in TCA (citric acid step. This is the only step in TCA (citric acid cycle) that involves cycle) that involves substrate-level substrate-level phosphoyrlationphosphoyrlation to directly produce a high to directly produce a high energy phosphate bond.energy phosphate bond.

REACTIONS contd.REACTIONS contd.6.6. SUUCINATE DEHYDROGENASE SYNTHETASE SUUCINATE DEHYDROGENASE SYNTHETASE

oxidizes succinate to oxidizes succinate to fumaratefumarate. This enzyme is . This enzyme is membrane bound in the mitochondria and membrane bound in the mitochondria and transfers two H+ to FAD to form FADHtransfers two H+ to FAD to form FADH2. 2. It is It is inhibited by oxaloacetate.inhibited by oxaloacetate.

7.7. FUMARATE HYDRATASE (fumarase) reversebly FUMARATE HYDRATASE (fumarase) reversebly hydrates fumarate to form hydrates fumarate to form malatemalate..

8.8. MALATE DEHYDRGENASE forms MALATE DEHYDRGENASE forms oxaloacetate oxaloacetate and one more FADH from malate to complete the and one more FADH from malate to complete the cyclecycle

Hence, one cycle produces 1 GTP (step 5), 3 Hence, one cycle produces 1 GTP (step 5), 3 NADH’s NADH’s

(steps 3 ,4, and 8) and 1FADH(steps 3 ,4, and 8) and 1FADH22 (step 6). (step 6).

SUMMARY OF THE REACTIONSSUMMARY OF THE REACTIONSThe sum of all reactions in the citric acid The sum of all reactions in the citric acid

cycle is:cycle is:Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 3 Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 3

H2O →H2O →CoA-SH + 3 NADH + H+ + FADH2 + GTP + CoA-SH + 3 NADH + H+ + FADH2 + GTP + 2 CO2 + 3 H+ 2 CO2 + 3 H+

Two carbons are Two carbons are oxidizedoxidized to CO2, and the to CO2, and the energy from these reactions is stored in energy from these reactions is stored in GTPGTP , NADH and FADH2. NADH and FADH2 , NADH and FADH2. NADH and FADH2 are are coenzymescoenzymes (molecules that enable or (molecules that enable or enhance enzymes) that store energy and enhance enzymes) that store energy and are utilized in oxidative phosphorylation.are utilized in oxidative phosphorylation.

SIMPLIFIED VIEW OF THE SIMPLIFIED VIEW OF THE PROCESSPROCESS The process begins with the oxidation of pyruvate, producing The process begins with the oxidation of pyruvate, producing

one CO2, and one acetyl-CoA. one CO2, and one acetyl-CoA.

Acetyl-CoA reacts with the four carbon carboxylic acid, Acetyl-CoA reacts with the four carbon carboxylic acid, oxaloacetate--to form the six carbon carboxylic acid, citrate. oxaloacetate--to form the six carbon carboxylic acid, citrate.

Through a series of reactions citrate is converted back to Through a series of reactions citrate is converted back to oxaloacetate. This cycle produces 2 CO2 and consumes 3 oxaloacetate. This cycle produces 2 CO2 and consumes 3 NAD+, producing 3 NADH and 3 H+. NAD+, producing 3 NADH and 3 H+.

It consumes 3 H2O and consumes one FAD, producing one It consumes 3 H2O and consumes one FAD, producing one FADH+. FADH+.

1st turn end= 1 ATP, 3 NADH, 1 FADH2, 2 CO2 1st turn end= 1 ATP, 3 NADH, 1 FADH2, 2 CO2

Since there are two molecules of Pyruvic acid to deal with, the Since there are two molecules of Pyruvic acid to deal with, the cycle turns once more. cycle turns once more.

The complete end result= 2 ATP, 6 NADH, 2 FADH2, 4 CO2 The complete end result= 2 ATP, 6 NADH, 2 FADH2, 4 CO2

REGULATIONREGULATION Many of the enzymes in the TCA cycle are regulated Many of the enzymes in the TCA cycle are regulated

by negative feedback from ATP when the energy by negative feedback from ATP when the energy charge of the cell is high. charge of the cell is high.

Such enzymes include the pyruvate dehydrogenase Such enzymes include the pyruvate dehydrogenase complex that synthesises the acetyl-CoA needed for complex that synthesises the acetyl-CoA needed for the first reaction of the TCA cycle.the first reaction of the TCA cycle.

Also the enzymes citrate synthase, isocitrate Also the enzymes citrate synthase, isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase and alpha-ketoglutarate dehydrogenase, that regulate the first three steps of dehydrogenase, that regulate the first three steps of the TCA cycle, are inhibited by high concentrations of the TCA cycle, are inhibited by high concentrations of ATP.ATP.

This regulation ensures that the TCA cycle will not This regulation ensures that the TCA cycle will not oxidise excessive amounts of pyruvate and acetyl-CoA oxidise excessive amounts of pyruvate and acetyl-CoA when ATP in the cell is plentiful. This type of negative when ATP in the cell is plentiful. This type of negative regulation by ATP is by an allosteric mechanismregulation by ATP is by an allosteric mechanism

REGULATION contd.REGULATION contd. Several enzymes are also negatively Several enzymes are also negatively

regulated when the level of reducing regulated when the level of reducing equivalents in a cell are high (high equivalents in a cell are high (high ratio of NADH/NAD+). ratio of NADH/NAD+).

This mechanism for regulation is due This mechanism for regulation is due to substrate inhibition by NADH of the to substrate inhibition by NADH of the enzymes that use NAD+ as a enzymes that use NAD+ as a substrate. substrate.

This includes both the entry point This includes both the entry point enzymes pyruvate dehydrogenase enzymes pyruvate dehydrogenase and citrate synthase.and citrate synthase.

ENERGETICS OF TCAENERGETICS OF TCA

The overall consumption of one molecule of The overall consumption of one molecule of acetyl-CoA in the TCA is a spontaneous, acetyl-CoA in the TCA is a spontaneous, exergonic process (having overall negative free exergonic process (having overall negative free energy change) ; energy change) ; ΔΔGGoo’ = -60 kJ mol’ = -60 kJ mol-1-1..

The rate of utilization of the ACoA in the cycle The rate of utilization of the ACoA in the cycle depends on the energy status within the depends on the energy status within the mitochondria.mitochondria.

Under conditions of high energy, the Under conditions of high energy, the concentrations of NADH and ATP are high, and concentrations of NADH and ATP are high, and those of NAD+ and FADH2 are low.those of NAD+ and FADH2 are low.

The reoxidation of NADH and FADH2 occurs in the The reoxidation of NADH and FADH2 occurs in the electron transport chain and is necessary for he electron transport chain and is necessary for he cycle to continue.cycle to continue.

IMPORTANT FACTSIMPORTANT FACTS

Many of the intermediates of the Many of the intermediates of the citric acid cycle are used in in the citric acid cycle are used in in the synthesis of other biomoleculessynthesis of other biomolecules

Many biomolecules feed into the Many biomolecules feed into the citric acid cyclecitric acid cycle

Thus, the TCA is considered to be Thus, the TCA is considered to be amphibolicamphibolic

OVERALL CHEMICAL OVERALL CHEMICAL REACTIONS DURING ONE REACTIONS DURING ONE TURN OF TCATURN OF TCA

The overall reactions are the The overall reactions are the complete complete oxidationoxidation of one of one molecule of ACoA, the molecule of ACoA, the release release of two moleculesof two molecules of CO2, the of CO2, the reductionreduction of three molecules of of three molecules of NAD+ and one FAD, and the NAD+ and one FAD, and the phosphorylationphosphorylation of one of one molecule of GDP.molecule of GDP.