Tricarboxylic Acid Cycle (TCA), Krebs Cycle

Tricarboxylic Acid Cycle (TCA), Krebs Cycle

Occurs totally in mitochondria Pyruvate (actually acetate) from

glycolysis is degraded to CO2 Some ATP is produced More NADH is made NADH goes on to make more ATP in

electron transport and oxidative phosphorylation

Traffic circle, comp. entering & leaving

Tricarboxylic Acid Cycle (TCA),

Oxidative Decarboxylation of Pyruvate

Pyr. from aerobic glycolysis is transported to cross inner mitochondrial membrane by specific transporter. In the matrix, pyr. is irreversibly decarboxylated by a multienzyme complex Five coenzyme’re needed See figure

Oxidative Decarboxylation of Pyruvate

Pyr is converted to acetyl CoA by pyr dehydrogenase (pyr DH) complex , which is a multienzyme complex.

pyr dehydrogenase complex is not part of TCA cycle proper, but is a mojor source of acetyl CoA.

The irreversibility of the reaction explains why glucose can not be formed from acetyl CoA in gluconeogenesis.

Oxidative Decarboxylation of Pyruvate

pyr dehydrogenase complex is composed of three enzymes – pyr decarboxylase (E1) - dihydrolipoyl transacylase (E2) - dihydrolipoyl dehydrogenase (E3)

Each catalyzed a part of the overall reaction In addition to two regulatory enzymes

protein kinase and phosphoprotein phosphatase.

Oxidative Decarboxylation of Pyruvate

Coenzymes: Pyr DH complex contains 5 coenzyme which act as a carriers or oxidant for intermediates.

(1) Thiamine pyrophosphate(2)Lipoic acid(3) CoA(4) FAD(5) NAD

Mechanism of Pyr. decarboxylase

Regulation of Pyr. Dehydrogenase

Complex Allosteric activation of kinase & Phosphatase:- Cyclic AMP-independent protein kinase

( activated)activates phosphorylated E1 ( inactive ) & inhibits dephosphorylated ( active ) inhibit Pyr DH.

- protein kinase allosterically activated by ATP, acetyl CoA, NADH ( high energy signals) inhibit Pyr DH (turned off).

- protein kinase allosterically inactivated by NAD+ CoA, ( low energy signals) activate Pyr DH (turned ).

- Pyr is a potent inhibitor of kinase, if pyr concentration is elevated so E1 is active

- Ca+ is strong activator of Phosphatase, stimulating E1 activity ( skeletat muscle contraction)

Regulation of Pyr. Dehydrogenase Complex

Reactions of TCA Synthesis of citrate from acetyl CoA and

oxaloacetate (OAA): Irreversible, catalyzed by citrate

synthase. Aldol condensation reaction. citrate synthase is inhibited by ATP,

NADH, succinyl CoA & fatty acyle CoA. Function of citrate: It provides a source

of acetyl CoA for fatty acid synthesis & it inhibits PFK1

Reactions of TCA (3) Isomerisation of citrate: to isocitrate by

aconitase ( reversible reaction), It is inhibited by fluroacetate, a compound used for rat poisoning(fluroacetate is converted to flurocitrate which is a potent inhibitor for aconitase)

(4) Oxidative Decarboxylation of isocitrate: irreversible oxidative phosphorylation, by isocitrate DH to give -Ketoglutarate, NADH & CO2

-It is rate limiting step-isocitrate DH is activated by ADP and Ca +2 &

inhibited by ATP, NADH

Reactions of TCA (5) Oxidative Decarboxylation of -

Ketoglutarate: by -Ketoglutarate DH to give succinyle CoA (similar to pyr DH),

Release of 2nd NADH & CO2

-Ketoglutarate DH need coenzymes TPP,NAD,FAD,CoA& lipoic acid.

-Ketoglutarate DH is inhibited by ATP,NADH, GTP& succinyle CoA. And activated by Ca +2 .

However it is not regulated by the phosphorylation and de phosphorylation reaction that describe in Pyr DH

Reactions of TCA (5) Cleavage of succinyle CoA: Cleavage of

(high-energy thioester dound) succinyle CoA to succinate by succinate thiokinase.

It is coupled by release of GTPwhich inter-converted by nucleoside diphosphate kinase reaction

Substrate –level phosphorylation. succinyle CoA can be produced from

Proponyle CoA ( metabolism of fatty acids)

Reactions of TCA

(6) Oxidation of succinate: to fumarate by succinate DH, producing FADH2

(7) Hydration of fumarate: to malate by fumarase

(8)Oxidation of malate: By malate DH

To OAA & 3nd NADH.

Regulation of TCA Cycle

Intermediates for Biosynthesis -Ketoglutarate is transaminated to

make glutamate, which can be used to make purine nucleotides, Arg and Pro

Succinyl-CoA can be used to make porphyrins

Fumarate and oxaloacetate can be used to make several amino acids and also pyrimidine nucleotides

mitochondrial citrate can be exported to be a cytoplasmic source of acetyl-CoA (F.A in fed state) and oxaloacetate glucose in fast state

Biosynthetic & Anaplerotic reactions

Anaplerotic Reactions (filling up reactions)

PEP carboxylase - converts PEP to oxaloacetate

Pyruvate carboxylase - converts pyruvate to oxaloacetate

Malic enzyme converts pyruvate to malate

See fig. Reactions from1-5 is anaplerotic i.e. filling up reactions

Membrane Transport System The inner mitochondrial membrane is

impermeable to the most charged and hydrophilic substances. However it contains numerous transport proteins that permit the passage of specific molecules.

1- ATP-ADP transport, see oxid-phospho, Transporter for ADP & Pi from cytosol into

mitochondria by specialized carriers ( adenine nucleotide carrier) which transport ADP from cytosol into mitochondria, while exporting ATP from matrix back into the cytosol .

Membrane Transport System Transport of reducing equivalents from cytosol

into mitochondria using: The inner mitochondrial membrane lacks an NADH transport proteins, NADH produced in cytosol cannot directly penetrate into mitochondria. However two electron of NADH ( called reducing equivalents) are transported by using shuttle.

1. glycerophosphate shuttle ( results in synthesis of 2 ATP for each cytosolic NADH oxidized )

2. malate-aspartate shuttle ( results in synthesis of 3 ATP in the mitochondrial matrix for each cytosolic NADH oxidized )

Membrane Transport System

Pyruvate DH deficiency. Pyruvate DH deficiency is the most

common biochemical cause of congenital lactic acidosis.

Pyruvate cannot to acetyl CoA but to lactate

The most sever form cause neonatal death.

The moderate form cause psychomotor retardation with damage in cerebral cortx, basal ganglia and brain stem and death.

The third form cause episodic ataxia.

Energy produced from TCA