Triacylcaboxylic Acid Cycle Kerb Cycle TCA Cycle By

Triacylcaboxylic Acid Cycle Kerb Cycle/ TCA Cycle By Tahir ASAB

Overview • • TCA cycle also called the Krebs cycle or the citric acid cycle, plays several roles in metabolism. It is final pathway where the oxidative metabolism of carbohydrates, amino acids, & fatty acids converge, their carbon skeletons being converted to CO 2. This oxidation provides energy for the production of the majority of ATP in most animals, including humans. The cycle totally occurs totally in the mitochondria & is, therefore, in close proximity to the reactions of electron transport, which oxidize the reduced coenzymes produced by the cycle. The TCA cycle is a an aerobic pathway, because O 2 is required as the final electron acceptor. The TCA cycle also participates in a number of important synthetic reaction e. g formation of glucose from the carbon skeletons of some amino acids, and it provides building blocks for the synthesis of some amino acid and heme. The cycle should not be viewed as a closed circle, but instead as a traffic circle with compounds entering & leaving as required. 3

The Citric Acid Cycle The citric acid cycle is the final common pathway for the oxidation of fuel molecules: amino acids, fatty acids, & carbohydrates. • Most fuel molecules enter the cycle as acetyl coenzyme A • This cycle is the central metabolic hub of the cell • It is the gateway to aerobic metabolism for any molecule that can be transformed into an acetyl group or dicarboxylic acid, • It is also an important source of precursors for building blocks • Also known as, Krebs Cycle, & Tricarboxylic Acid Cycle (TCA) ØThe citric acid cycle oxidizes two-carbon units ØEntry to the cycle and metabolism through it are controlled ØThe cycle is a source of biosynthetic precursors 4

Introduction 1. 2. 3. 4. 5. 6. 7. The function of the cycle is the harvesting of high-energy electrons from carbon fuels The cycle itself neither generates ATP nor includes O 2 as a reactant Instead, it removes electrons from acetyl Co. A & uses them to form NADH & FADH 2 (high-energy electron carriers) In oxidative phosphorylation, electrons from reoxidation of NADH & FADH 2 flow through a series of membrane proteins (electron transport chain) to generate a proton gradient These protons then flow back through ATP synthase to generate ATP from ADP & inorganic phosphate O 2 is the final electron acceptor at the end of the electron transport chain The cytric acid cycle + oxidative phosphorylation provide > 95% of energy used in human aerobic cells 5

Figure 9. 1 The tricarboxylic acid cycle shown as a part of the central pathways of energy metabolism.

A single molecule of glucose can potentially yield ~38 molecules of ATP 7

Fuel for the Citric Acid Cycle Initiates cycle Pantothenate Thioester bond to acetate -mercapto-ethylamine 8

Mitochondrion Double membrane, & cristae: invaginations of inner membrane 9

Overview of inter-relationship of glycolysis, pyruvate carboxylase, citric acid cycle, proton pumps and ATP-synthase. 10

What do mitochondria look like? Classic View: discrete structures (sausage-like) New View: interconnected structures floating in the cytosol 11

The DNA found in mitochondria is entirely different from the DNA found in the nucleus and indicates that mitochondria probably evolved from a type of bacteria, amino acid sequence of the ATP synthase is well conserved across eukaryotes and prokaryotes. 12

Mitochondrion Oxidative decarboxilation of pyruvate, & citric acid cycle take place in matrix, along with fatty acid oxidation Site of oxidative phosphorylation Permeable 13

Citric Acid Cycle: Overview Input: 2 -carbon units Output: 2 CO 2, 1 GTP, & 8 high-energy electrons 14

Cellular Respiration 8 high-energy electrons from carbon fuels Electrons reduce O 2 to generate a proton gradient ATP synthesized from proton gradient 15

Glycolysis to citric acid cycle link Acetyl Co. A link is the fuel for the citric acid cycle 16

Pyurvate dehydrogenase complex A large, highly integrated complex of three kinds of enzymes Pyruvate + Co. A + NAD+ acetyl Co. A + CO 2 + NADH Groups travel from one active site to another, connected by tethers to the core of the structure 17

Figure 9. 2 Oxidative decarboxylation of pyruvate. Product inhibition is shown, but covalent modification is the key method of regulation for PDH is active when dephosphorylated.

Figure 9. 3 Mechanism of action of the pyruvate dehydrogenase complex. TPP = thiamine pyrophosphate; L = lipoic acid.

Figure 9. 4 Regulation of pyruvate dehydrogenase complex.

Figure 9. 5 means of regulation for citrate synthase. The binding of oxaloacetate Formation of α-ketoglutarate from causes a conformational change in the enzyme that generates a acetyl Co. A and oxaloacetate.

Figure 9. 6 Formation of malate from α-ketoglutarate.

Figure 9. 7 Formation of oxaloacetate from malate.

The citric acid cycle 24

Tricarboxylic Acid Cycle (TCA/Krebs Cycle) is the CENTRAL HUB for oxidation and energy production from sugars, fatty acids, and some amino acids! TCA is called a “cycle” because the last step creates the substrate for the first step! Acetyl-Co. A is main entry molecule! • Glucose Acetyl-Co. A • Fatty Acids Acetyl-Co. A • Amino Acids Some make Acetyl-Co. A Some aa turned into Glucose ( Acetyl-Co. A) Complete Oxidation of one Acetyl-Co. A GTP+3 NADH+FADH 2+2 CO 2 • GTP ATP • NADH 3 ATP • FADH 2 2 ATP Acetyl-Co. A 1 ATP+9 ATP+2 CO 2 25

Energy Balance Sheet for complete oxidation of a single glucose molecule to carbon dioxide. • Glycolysis in Cytosol: (8 ATP) – – • 1 glucose 2 pyruvate + 2 ATP + 2 NADH System feeds forward only if NAD+ is available Aerobic and sometimes anaerobic Pyruvate transported into matrix Pyruvate Decarboxylase in Matrix: (6 ATP) 2 pyruvate 2 Acetyl-Co. A+2 NADH + 2 H+ and 2 CO 2 • TCA in Matrix: (24 ATP) – 2 Acetyl-Co. A 4 CO 2+2 GTP+2 FADH 2+6 NADH – Conversion to ATP 2 ATP + 4 ATP + 18 ATP • • Net Yield: 1 glucose 8+6+24=38 ATP (assumes oxygen ) Sometimes a less efficient system is used to transport cytosolic NADH into the mitochondrial matrix such that these two NADHs yield only 4 ATP, not 6. 26

Figure 9. 8 Number of ATP molecules produced from the oxidation of one molecule of acetyl Co. A (using both substrate-level and oxidative phosphorylation).

Figure 9. 9 A. Production of reduced coenzymes, ATP, and CO 2 in the citric acid cycle.

Pyruvate to Acetyl Co. A, irreversible Key irreversible step in the metabolism of glucose 29

Regulation of CAC: Rate controlling enzymes: Citrate synthatase Isocitrate dehydrogenase a-keoglutaratedehydrogenase Regulation of activity by: Substrate availability Product inhibition Allosteric inhibition or activation by other intermediates 30

Regulation of pyruvate dehydrogenase Inhibited by products, NADH & Acetyl Co. A Also regulated by covalent modification, the kinase & phosphatase also regulated 31

Control of citric acid cycle Regulated primarily by ATP & NADH concentrations, control points: isocitrate dehydrogenase & - ketoglutarate dehydrogenase (citrate synthase - in bacteria) 32

Biosynthetic roles of the citric acid cycle 33

Arsenic Compound poisoning: Inactivation of E-2 of PDC, and other proteins. Organic Arsenical were used as antibiotics for the treatment of syphilis and trypanosomiasis. Micro-organisms are more sensitive to organic arsenicals than humans. But these compounds had severe side effects and Aspoisoning. Fowler’s solution, the famous 19 th century tonic contained 10 mg/ml As. Charles Darwin died of As poisoning by taking this tonic. Napoleon Bonaparte’s death was also suspected to be due to As poisoning. 34

Summary: What does the electron transport pathway look like? NADH and FADH 2 feed e- into system via complex I OR II of the inner mitochondrial membrane depending on how much energy they contain. 35

Why do amino acids make a poor fuel for making ATP? Answer: It is really expensive AND potentially toxic! • 1) Use results in protein breakdown!! expensive • 2) Not all A. A. can feed into glucose or the TCA!! expensive • 3) Ammonia and urea are created by degradation!! toxic • 4) Ketones are created by accident!! Toxic • During Starvation: Proteolysis occurs in liver cells so glucose can be produced for the other cells that MUST use glucose, like glucose dependent red blood cells. • These are “gluconeogenic” amino acids! • Some amino acids “can” feed into TCA following transamination! 36

Flavine. Adenine. Dinucleotide has a triple-ringed flavin with alternating double bonds that temporarily hold (stabilize) electrons. 37

Thiamin (Vitamine B 1) deficiency causes Beriberi: Thiamine pyrophosphate (TPP) is an important cofactor of pyruvate dehydrogenase complex, or PDC a critical enzyme in glucose metabolism. Thiamine is neither synthesized nor stored in good amounts by most vertebrates. It is required in the diets of most vertebrates. Thiamine deficiency ultimately causes a fatal disease called Beriberi characterized by neurological disturbances, paralysis, atrophy of limbs and cardiac failure. Note that brain exclusively uses aerobic glucose catabolism for energy and PDC is very critical for aerobic catabolism. Therefore thiamine deficiency causes severe neurological symptoms. Arsenic Poisoning: Arsenic compounds such as arsenite (As. O 3 ---) organic arsenicals are poisonous because they covalently bind to sulfhydryl compounds (SH- groups of proteins and cofactors). Dihydrolipoamide is a critical cofactor of PDC, and it has two-SH groups, which are important for the PDC reaction. These –SH groups are covalently inactivated by arsenic compounds as shown below; OH -O As HS S + OH -O HS As + 2 H 2 O S R R 38

Arsenic compounds in low doses are very toxic to microorganisms, therefore these compounds were used for the treatment of syphilis and other diseases in earlier days. Arsenicals were first antibiotics, but with a terrible side effects as they are eventually very toxic to humans. Unfortunately and ignorantly, a common nineteenth century tonic, the Fowler’s solution contained 10 mg/ml arsenite. This tonic must have been responsible for many deaths, including the death of the famous evolution scientist Charlse Darwin. 39