Hans Adolf Krebs :Biochemist; born in Germany. Worked in Britain. His discovery in 1937 of the “KREBS CYCLE” of chemical reaction was critical to the understanding of cell metabolism and earned him Nobel Prize for Physiology or medicine. He discovered CAC in Pigeon Flight Muscle

CITRIC ACID CYCLE

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Definition Location Source and fate of Acetyl CoA Reactions Regulation Energetics Significance Inhibition

DEFINITION:

The citric acid cycle is a series of reactions that brings about catabolism of acetyl-coA liberating reducing equivalents which upon oxidation through respiratory chain of mitochondria, generate ATP.

It plays a central role in the breakdown or catabolism of organic fuel molecules—i.e glucose and some other sugars, fatty acids, and some amino acids. Before these rather large molecules can enter the TCA cycle they must be degraded into a two-carbon compound called acetyl coenzyme A (acetyl CoA). Once fed into the TCA cycle, acetyl CoA is converted into carbon

dioxide  and energy.

The enzymes of TCA in mitochondrial matrix present either free or attached to surface of inner mitochondrial membrane.

WHY TCA IS CALLED AMPHIBOLIC?

It plays both catabolic and anabolic role.

*Catabolic role:

Acetyl CoA is oxidized to C02, H2O giving out energy.

*Anabolic role:

Intermediates of TCA cycle plays a role in synthesis like heme formation, FA synthesis, Cholesterol, Steroid synthesis.

Step 1 : condensation

Step 2 : dehydration/Rehydration

Step 3: Oxidative decarboxylation

Step 4: Oxidative decarboxylation

Step 5: Phosphorylation

Step 6:Dehyrogenation

Step 7:Hydration

Step 8: Dehydrogenation

REACTIONS:

Oxidative decarboxylation of pyruvate to acetyl CoA by pyruvate dehydrogenase complex occurs .This step is a connecting link between glycolysis and TCA cycle.

1. Formation of citrate : Krebs cycle starts with the condensation of acetyl CoA and oxaloacetate catalysed by the enzyme citrate synthase.

2.Citrate is converted to Isocitrate by enzyme aconitase. This takes place in two steps:

- dehydration to cis-aconitase

-rehydration to Isocitrate.

3. Formation of a-Ketoglutarate: Isocitrate undergoes dehydrogenation by Isocitrate dehydrogenase to form Oxalosuccinate. There follows decarboxylation to α-Ketoglutarate which also catalyzed by Isocitrate dehydrogenase.

Formation of NADH and liberation of CO2 occur.

The three different forms of enzyme are:

a. One which is NAD+ dependent – found in mitochondria.

b. Other two enzymes are NADP+ dependent – found in mitochondria and cytosol.

4. Conversion of a-Ketoglutarate to Succinyl CoA : α-Ketoglutarate undergo oxidative decarboxylation catalyzed by α-Ketoglutarate dehydrogenase to give Succinyl –CoA. This enzyme is dependent on five cofactors-TPP, lipoamide, NAD+, FAD and CoA.

Second NADH is produced with liberation of CO2.

5. Formation of succinate : Succinyl CoA is converted to succinate by succinate thiokinase.

This reaction is coupled with the phosphorylation of GDP to GTP. This is a substrate level phosphorylation.

6. Conversion of succinate to fumarate : Succinate is oxidized by succinate dehydrogenase

to fumarate. This reaction results in the production of FADH2.

7.Formation of malate : The enzyme fumarase catalyses the conversion of fumarate to malate with the addition of H2O.

8. Conversion of malate to oxaloacetate : Malate is then oxidized to oxaloacetate by malate dehydrogenase. The third and final synthesis of NADH occurs at this stage. The oxaloacetate is regenerated which can combine with another molecule of acetyl CoA and continue the cycle.

AA FORMING COMPOUNDS

*Pyruvate:Glycine, Alanine, Serine, Cysteine, Threonine, Hydroxy-proline

and tryptophan.*a-Ketoglutarate :

Arginine, Histidine, Glutamine and Proline.*Fumarate :

Phenylalanine, Tyrosine.*Succinyl-CoA :

Valine, Methionine, Isoleucine.

ENERGETICS OF CITRIC ACID CYCLE :

During the process of oxidation of acetyl-CoA via citric acid cycle, 4 reducing equivalents (3 as NADH and one as FADH2) are produced. Oxidation of 3 NADH by electron transport chain coupled with oxidative

phosphorylation results in the synthesis of 9 ATP. FADH2 leads to the formation of 2 ATP. Besides there is one substrate level

phosphorylation. Thus a total of TWELVE ATP are produced from one acetyl- CoA.

REGULATION :

TCA cycle is regulated at four step : Pyruvate dehydrogenase citrate synthase isocitrate dehydrogenase a-Ketoglutarate dehydrogenase

1. Citrate synthase is inhibited by ATP, NADH, acetyl CoA and Succinyl CoA.

2. Isocitrate dehydrogenase is activated by ADP and inhibited by ATP and NADH.

3. a-Ketoglutarate dehydrogenase is inhibited by Succinyl CoA and NADH.

4. Availability of ADP is very important for the citric acid cycle to proceed. This is due to the fact that unless sufficient levels of ADP are available, oxidation (coupled with phosphorylation of ADP to ATP) of NADH and FADH2 through electron transport chain stops.

The accumulation of NADH and FADH2 will lead to inhibition of the enzymes and also limits the supply of NAD+ and FAD which are essential for TCA cycle to proceed.

SIGNIFICANCE: To provide energy in the form of ATP. To Provide substrate for the respiratory chain. Final common pathway for oxidation of carbohydrate, lipids, protein as

glucose, fatty acids and many amino acids are all metabolized to acetyl-CoA or intermediates of the cycle.

Role in synthesis of nonessential amino acids :Since the transamination reactions are reversible, the cycle also serves as a source of carbon skeletons for the synthesis of some amino acids like Alanine, aspartate, Asparagine Glutamate , glutamine etc

Role in fatty acid synthesis: Acetyl-CoA, formed from pyruvate by the action of pyruvate dehydrogenase, is the major substrate for long-chain fatty acid synthesis .

Role in Heme synthesis : Succinyl CoA condenses with amino acid Glycine to form Alpha amino beta keto Adipic acid, which is the first step of haem biosynthesis.

Role in purine and pyrimidine synthesis : Glutamate and Aspartate derived from TCA cycle are utilized for the synthesis of purines and pyrimidine

Four water soluble vitamins of B- complex ( Riboflavin, Niacin , Thiamine , Pantothenic acid ) have a precise role .

VITAMIN COENZYME FOR

1.Riboflavin a-Ketoglutarate dehydrogenase , succinate dehydrogenase

2.Niacin a-Ketoglutarate dehydrogenase, Isocitrate dehydrogenase, malate dehydrogenase.

3.Thiamine a-Ketoglutarate dehydrogenase

4.Pantothenic acid Cofactor attached to active carboxylic acid residue like acetyl CoA,Succinyl CoA

INHIBITORS OF KREBS CYCLE

The important enzymes of TCA cycle inhibited by the respective inhibitors are listed :

Enzyme Inhibitor1. Aconitase Fluoroacetate (Non- competitive)2. a-Ketoglutarate dehydrogenase Arsenite (Non- competitive)3. Succinate dehydrogenase Malonate (Competitive)