Overview of Metabolism &

The Citric acid cycle

Dr.S.Chakravarty M.D.

Carbohydrates

Fats

Proteins

recycling

3 Stages Of Metabolism

1

2

3

Definition of TCA cycle

The citric acid cycle (Krebs cycle, tricarboxylic acid cycle) is a sequence of reactions in mitochondria that oxidizes the acetyl moiety of acetyl-CoA and reduces coenzymes that are reoxidized through the electron transport chain, linked to the formation of ATP.

STEP 1 PYRUVATE to Acetyl CoA

Enzyme :- Pyruvate Dehydrogenase Complex

ADVANTAGES OF A MULTI ENZYME COMPLEX :-

– RAPID TRANSFER OF INTERMEDIATES IN BETWEEN ACTIVE SITES OF INDIVIDUAL ENZYMES ENHANCING REACTION RATE

– MINIMIZES SIDE REACTIONS BY CHANNELING INTO A SINGLE PATHWAY AND DECREASING OUTSIDE REACTIONS .

– EFFECTIVE CONTROL AND COORDINATION OF THE REACTIONS BETTER METABOLIC CONTROL

Site :- Inner mitochondrial membrane

How does Pyruvate enter mitochondria?

• Symport along with H+ ions

• E1 - Thiamine pyro phosphate (TPP) (B1) • E2 – Lipoic acid Co-enzyme-A – (Pantothenic acid)

• E3 – NAD – Niacin (B3) FAD – Riboflavin (B2)

The Enzyme subunits

Tender – Thiamine Loving - LipoamideCare -CoASHFor – (FAD)RiboflavinNancy- NAD (Niacin)

3 Enzymes:- 1)PDH,

2)α-KGDH(TCA cycle)

3)Branched keto acid dehydrogenase

The Coenzymes and Prosthetic Groups of Pyruvate Dehydrogenase

Cofactor Location Function

TPP(Thiamine pyrophosphate)

Bound to E1 Decarboxylates pyruvate yielding a hydroxyethyl TPP carbanion

Lipoic acid Covalently linked to a Lys on E2(lipoamide)

Accepts the hydroxyethyl carbanion from TPP as an acetyl group

Co A( Coenzyme A) Substrate for E2 Accepts acetyl group from lipoamide

FAD(Flavin adenine dinucleotide)

Bound to EE3 Reduced by lipoamide

NAD+ Substrate for E3 Reduced by FADH2

IMPORTANCE

• PDH is IRREVERSIBLE ( Fats cannot be converted to glucose.)

• COMMITTED STEP in oxidation of glucose.

• ENERGETICS :- 1 NADH IS GENERATED = 2.5 ATP

• REGULATION :- – End product as well as covalent modification– Phosphorylation of enzyme by a kinase decreases the activity and

dephosphorylation increases the activity.

Regulation of PDH enzyme:

1. Regulation by end product inhibition (Allosteric)

2. Regulation by Covalent modification:

• PDH kinase – inactivation of enzyme

• PDH Phosphatase - activation

Congenital Lactic acidosis:

• Deficiency of Pyruvate Dehydrogenase enzyme.

• Inability to convert Pyruvate to Acetyl co-A.

• Shunted to Lactate Dehydrogenase to form Lactic Acid.

• Deficient NADH leading to deficient ATP

• Lactic acidosis, severe psychomotor retardation, damage to brain stem, cortex etc.

Type: Reasons:

Other causes of lactic acidosis:

• Mercury poisoning• Arsenic poisoning

• Pyruvate carboxylase deficiency

• TPP deficiency

• Chronic Alcoholism

• Binds to –SH groups of Lipoic acid and forms a stable complex.

• Decreased absorption and poor diet.

• Severe exercise excess lactate

Biomedical Importance• The citric acid cycle is the final common pathway for the

oxidation of carbohydrate, lipid, and protein because glucose, fatty acids, and most amino acids are metabolized to acetyl-CoA or intermediates of the cycle.

• It is a source of reduced co-enzymes that provide the substrates for the respiratory chain.

• It is both catabolic and anabolic (amphibolic).

contd..

Biomedical Importance• It also has a central role in gluconeogenesis, lipogenesis, and

interconversion of amino acids. – So, components of the cycle have a direct or indirect controlling effects in key

enzymes of other pathways.

• Many of these processes occur in most tissues, but the liver is the only tissue in which all occur to a significant extent. – The repercussions are therefore profound when, for example, large numbers

of hepatic cells are damaged as in acute hepatitis or replaced by connective tissue (as in cirrhosis).

• Very few, if any, genetic abnormalities of citric acid cycle enzymes have been reported; such abnormalities would be incompatible with life or normal development.

The final common pathway

Sites

• Tissues :- All tissues

• Subcellular site :- Mitochondrial Matrix

Enzyme bound

Enzyme bound

The Citric Acid Cycle

ATPSubstrate level

phosphorylation

NADH

NADH

FADH2

NADH

Thiamin, lipoate , FAD

Citrate Synthase- THE SODA POP and the world’s food Supply ?

• Cirtate – fruity flavour – used commercially in soft drinks .• PLASTICIZER and FOAM INHIBITOR • INDUSTRIALLY PRODUCED using fungus Aspergillus Niger

• ALUMINIUM(Al +3) ions –MOST ABUNDANT METAL IN EARTH’S CRUST-extremely toxic to PLANTS

PLANTS SECRETE CITRATE INTO THE SOIL WHICH CLELATES Al+3

Genetically engineered plants which secrete 5-6 times normal levels of citrate in soil ONE PROBABLE SOLUTION TO INCREASE FOOD PRODUCTION

Complete oxidation of Acetyl CoA

OAA is viewed as a catalyst , which enters into the cycle , causes complete oxidation of acetyl CoA , and is regenerated in the end without any loss.

Formation of ATPReaction catalyzed by Method of production ATP molecules formed

Isocitrate dehydrogenase Respiratory chain oxidation of NADH

2.5 (3)

-ketoglutarate dehydrogenase

Respiratory chain oxidation of NADH

2.5 (3)

Succinate thiokinase Substrate level phosphorylation

1

Succinate dehydrogenase Respiratory chain oxidation of FADH2

1.5(2)

Malate dehydrogenase Respiratory chain oxidation of NADH

2.5 (3)

Net –> 10 (12)

Significance of the cycle

1)Final common oxidative pathway

2)Fat is burnt on the wick of carbohydrates • Oxidation of fats need the help of Oxaloacetate

which enters into the cycle and is regenerated in the end .

• The major source of OAA is Pyruvate. (Carbohydrate)

3) Excess carbohydrates are converted to neutral fats via citrate and ATP-citrate lyase but not vice versa because Pyruvate dehydrogenase step is irreversible.

T

IRREVERSIBLE

P

4)Amphibolic ( Catabolic and Anabolic )

GABA

HEME

FATTY ACIDS,

STEROLSgluconeogenesis

5) TCA cycle plays an important role in Gluconeogenesis , Transmination and Deamination.

6) Anaplerotic ( filling – up) reactions -> As shown before ,TCA cycle acts as precursors of biosynthetic pathways , e.g Heme .

So, there is constant efflux of carbon units from the cycle .To counterbalance the loss , filling up reactions are necessary .

Eg. - Pyruvate to Oxaloacetate(PYRUVATE CARBOXYLASE) ( most important)

-- Phosphoenolpyruvate to Oxaloacetate (PEP CARBOXYLASE) --Pyruvate to Malate (Malic enzyme)

7) Metabolic traffic regulator -All metabolisms end in TCA. -Availability or lack of intermediates govern the directions of

pathways converging or going out of TCA.

Regulation of Citric Acid cycle • Regulation of the Citric Acid Cycle Depends Primarily on a Supply of

Oxidized Cofactors.

• Individual enzymes of the cycle are regulated - The most likely sites for regulation are the nonequilibrium reactions catalyzed by pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and -ketoglutarate dehydrogenase. The dehydrogenases are activated by Ca2+, which increases in concentration during muscular contraction and secretion, when there is increased energy demand.

contd ..

Regulation of Citric Acid cycle

• --Increased [ATP]/[ADP] and [NADH]/[NAD+] ratios inhibit PDH and the first three reactions.

• There is allosteric inhibition of citrate synthase by ATP and long-chain fatty acyl-CoA.

The availability of oxaloacetate, as controlled by malate dehydrogenase, depends on the [NADH]/[NAD+] ratio.

The concentration of oxaloacetate controls the rate of citrate formation.

Regulation of Iso-Citrate dehydrogenase:

Isocitrate Dehydrogenase

ATP

NADH

(-)

(-) Iso-citrate

Alpha ketoglutarate

(-)

Accumulation of Citrate

In well fed state:Inhibit Glycolysis

Enters fatty acid synthesis

In well fed state, increase in ATP and NADH will inhibit isocitrate dehydrogenase leading to accumulation of citrate. citrate will enter cytosol and inhibit Glycolysis and activates fatty acid synthesis.

Regulation of citric acid cycle

• Citrate synthase

• Iso-citrate dehydrogenase

• Alpha keto glutarate dehydrogenase

Inhibitors of TCA cycle

• Aconitase – is inhibited by fluoroacetate (non-competitive inhibition)

• -ketoglutarate dehydrogenase is inhibited by Arsenite (non-competitive inhibition)

• Succinate dehydrogenase is inhibited by Malonate (competitive inhibition)

Disorders related to TCA cycle• Beriberi , Wernicke’s encephalopathy and Korsakoff’s psychosis (WK

syndrome)in Thiamine deficiency is due to failure of TCA cycle ( Pyruvate dehydrogenase and - ketoglutarate dehydrogenase)

• Symptoms: confabulation, nystagmus (ophthalmoplegia), ataxia

• Congenital deficiency of Pyruvate dehydrogenase – Lactic acidosis and neurodeficit.

• Congenital deficiency of Pyruvate carboxylase – OAA is deficient – failure of sparking of TCA – severe mental retardation , lactic acidosis, hypoglycemia

• TCA cycle enzyme deficiencies are extremely rare.

• During a myocardial infarction , the oxygen supply to an area of the heart is dramatiocally reduced , forcing the cardiac myocytes to switch to anaerobic metabolism.Under these conditions , which of the following enzymes would be activated by increasing intracellular AMP?

A. Succinate dehydrogenaseB. PFK1C. GLUCOKINASE D. PDHE. LDH

Which of the following is required for cholesterol synthesis in hepatocytes?

• A. Citrate shuttle• B. Glycerphosphate shuttle• C. Malate-Aspartate shuttle• D. Carnitine shuttle• E. Adenine nucleotide shuttle

• A 55 year old alcoholic was brought to the emergency department by his friends. During their usual nightly gathering at the local bar, he had passed out and they had been unable to revive him.

• The physician ordered an injection of thiamine followed by overnight parental glucose. The next morning the patient was alert and serum thiamine was normal and blood glucose was 73mg/dl.

• The IV line was removed and he was taken home. At the time of discharge from hospital which of the following proteins would have no significant physiological activity in this patient?

– Malate dehydrogenase– Glucokinase– GLUT 1 transporter– PFK-1– Glucose 6 PO4 dehydrogenase

Thank you