Tricarboxylic Acid Cycle (TCA), Krebs Cycle Occurs totally in mitochondria Pyruvate (actually acetate) from glycolysis is degraded to CO 2 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 (E 1 ) - dihydrolipoyl transacylase (E 2 ) - dihydrolipoyl dehydrogenase (E 3 ) 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 E 1 ( 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 E 1 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 2 nd NADH & CO 2  -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 FADH 2 (7) Hydration of fumarate: to malate by fumarase (8)Oxidation of malate: By malate DH To OAA & 3 nd 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