Oxidative decarboxylation of pyruvate and Krebs cycle

OXIDATIVE DECARBOXYLATION OF PYRUVATE Matrix of the mitochondria contains pyruvate dehydrogenase complex

The fate of glucose molecule in the cell Synthesis of glycogen Glucose Pentose phosphate pathway Glycogen Glucose-6-phosphate Ribose, NADPH Degradation of glycogen Gluconeogenesis Glycolysis Ethanol Pyruvate Lactate Acetyl Co A

OXIDATIVE DECARBOXYLATION OF PYRUVATE Only about 7 % of the total potential energy present in glucose is released in glycolysis. Glycolysis is preliminary phase, preparing glucose for entry into aerobic metabolism. Pyruvate formed in the aerobic conditions undergoes conversion to acetyl CoA by pyruvate dehydrogenase complex.

OXIDATIVE DECARBOXYLATION OF PYRUVATE Pyruvate dehydrogenase complex is a bridge between glycolysis and aerobic metabolism – citric acid cycle Pyruvate dehydrogenase complex and enzymes of cytric acid cycle are located in the matrix of mitochondria 5

Entry of Pyruvate into the Mitochondrion Pyruvate freely diffuses through the outer membrane of mitochon-dria through the channels formed by transmembrane proteins porins. Pyruvate translocase, protein embedded into the inner membrane, transports pyruvate from the intermembrane space into the matrix in symport with H+ and exchange (antiport) for OH-.

Conversion of Pyruvate to Acetyl CoA Pyruvate dehydrogenase complex (PDH complex) is a multienzyme complex containing 3 enzymes, 5 coenzymes and other proteins. Pyruvate dehydrogenase complex is giant, with molecular mass ranging from 4 to 10 million daltons. Electron micrograph of the pyruvate dehydrogenase complex from E. coli.

Pyruvate dehydrogenase complex Enzymes: E1 = pyruvate dehydrogenase E2 = dihydrolipoyl acetyltransferase E3 = dihydrolipoyl dehydrogenase

Pyruvate dehydrogenase complex Coenzymes: TPP (thiamine pyrophosphate), lipoamide, HS-CoA, FAD+, NAD+. TPP is a prosthetic group of E1; lipoamide is a prosthetic group of E2; and FAD is a prosthetic group of E3. The building block of TPP is vitamin B1 (thiamin); NAD – vitamin B5 (nicotinamide); FAD – vitamin B2 (riboflavin), HS-CoA – vitamin B3 (pantothenic acid), lipoamide – lipoic acid 9

Overall reaction of pyruvate dehydrogenase complex Pyruvate dehydrogenase complex is a classic example of multienzyme complex Overall reaction of pyruvate dehydrogenase complex The oxidative decarboxylation of pyruvate catalized by pyruvate dehydrogenase complex occurs in five steps.

The Citric Acid Cycle Aerobic cells use a metabolic wheel – the citric acid cycle – to generate energy by acetyl CoA oxidation

Glucose Fatty Acids Amino Acids Synthesis of glycogen Glucose Pentose phosphate pathway Glycogen Glucose-6-phosphate Ribose, NADPH Degradation of glycogen Gluconeogenesis Glycolysis Ethanol Pyruvate Lactate Fatty Acids Amino Acids Acetyl Co A The citric acid cycle is the final common pathway for the oxidation of fuel molecules — amino acids, fatty acids, and carbohydrates. Most fuel molecules enter the cycle as acetyl coenzyme A.

Names: The Citric Acid Cycle Tricarboxylic Acid Cycle Krebs Cycle Hans Adolf Krebs. Biochemist; born in Germany. Worked in Britain. His discovery in 1937 of the ‘Krebs cycle’ of chemical reactions was critical to the understanding of cell metabolism and earned him the 1953 Nobel Prize for Physiology or Medicine. In eukaryotes the reactions of the citric acid cycle take place inside mitochondria

An Overview of the Citric Acid Cycle A four-carbon oxaloacetate condenses with a two-carbon acetyl unit to yield a six-carbon citrate. An isomer of citrate is oxidatively decarboxylated and five-carbon -ketoglutarate is formed. -ketoglutarate is oxidatively decarboxylated to yield a four-carbon succinate. Oxaloacetate is then regenerated from succinate.

An Overview of the Citric Acid Cycle Two carbon atoms (acetyl CoA) enter the cycle and two carbon atoms leave the cycle in the form of two molecules of carbon dioxide. Three hydride ions (six electrons) are transferred to three molecules of NAD+, one pair of hydrogen atoms (two electrons) is transferred to one molecule of FAD. The function of the citric acid cycle is the harvesting of high-energy electrons from acetyl CoA. 15

1. Citrate Synthase Citrate formed from acetyl CoA and oxaloacetate Only cycle reaction with C-C bond formation Addition of C2 unit (acetyl) to the keto double bond of C4 acid, oxaloacetate, to produce C6 compound, citrate citrate synthase

2. Aconitase Elimination of H2O from citrate to form C=C bond of cis-aconitate Stereospecific addition of H2O to cis-aconitate to form isocitrate aconitase

3. Isocitrate Dehydrogenase Oxidative decarboxylation of isocitrate to a-ketoglutarate (a metabolically irreversible reaction) One of four oxidation-reduction reactions of the cycle Hydride ion from the C-2 of isocitrate is transferred to NAD+ to form NADH Oxalosuccinate is decarboxylated to a-ketoglutarate isocitrate dehydrogenase

4. The -Ketoglutarate Dehydrogenase Complex Similar to pyruvate dehydrogenase complex Same coenzymes, identical mechanisms E1 - a-ketoglutarate dehydrogenase (with TPP) E2 – dihydrolipoyl succinyltransferase (with flexible lipoamide prosthetic group) E3 - dihydrolipoyl dehydrogenase (with FAD) a-ketoglutarate dehydrogenase

5. Succinyl-CoA Synthetase Free energy in thioester bond of succinyl CoA is conserved as GTP or ATP in higher animals (or ATP in plants, some bacteria) Substrate level phosphorylation reaction HS- + GTP + ADP GDP + ATP Succinyl-CoA Synthetase

6. The Succinate Dehydrogenase Complex Complex of several polypeptides, an FAD prosthetic group and iron-sulfur clusters Embedded in the inner mitochondrial membrane Electrons are transferred from succinate to FAD and then to ubiquinone (Q) in electron transport chain Dehydrogenation is stereospecific; only the trans isomer is formed Succinate Dehydrogenase

7. Fumarase Stereospecific trans addition of water to the double bond of fumarate to form L-malate Only the L isomer of malate is formed Fumarase

8. Malate Dehydrogenase Malate is oxidized to form oxaloacetate.

Stoichiometry of the Citric Acid Cycle Two carbon atoms enter the cycle in the form of acetyl CoA. Two carbon atoms leave the cycle in the form of CO2 . Four pairs of hydrogen atoms leave the cycle in four oxidation reactions (three molecules of NAD+ one molecule of FAD are reduced). One molecule of GTP, is formed. Two molecules of water are consumed.

Stoichiometry of the Citric Acid Cycle 9 ATP (2.5 ATP per NADH, and 1.5 ATP per FADH2) are produced during oxidative phosphorylation 1 ATP is directly formed in the citric acid cycle 1 acetyl CoA generates approximately 10 molecules of ATP 25

Functions of the Citric Acid Cycle Integration of metabolism. The citric acid cycle is amphibolic (both catabolic and anabolic). The cycle is involved in the aerobic catabolism of carbohydrates, lipids and amino acids. Intermediates of the cycle are starting points for many anabolic reactions. Yields energy in the form of GTP (ATP). Yields reducing power in the form of NADH2 and FADH2.

Regulation of the Citric Acid Cycle Pathway controlled by: (1) Allosteric modulators (2) Covalent modification of cycle enzymes (3) Supply of acetyl CoA (pyruvate dehydrogenase complex)

Regulation of the Citric Acid Cycle Three enzymes have regulatory properties citrate synthase (is allosterically inhibited by NADH, ATP, succinyl CoA, citrate – feedback inhibition) isocitrate dehydrogenase (allosteric effectors: (+) ADP; (-) NADH, ATP. Bacterial ICDH can be covalently modified by kinase/phosphatase) -ketoglutarate dehydrogenase complex (inhibition by ATP, succinyl CoA and NADH 28

Regulation of the citric acid cycle NADH, ATP, succinyl CoA, citrate -

Krebs Cycle is a Source of Biosynthetic Precursors Phosphoenol- pyruvate Glucose The citric acid cycle provides intermediates for biosyntheses

Role of the citric cycle in anabolism