Biochemistry department The Citric Acid Cycle Dr. Sooad Al-Daihan Biochemistry department

Introduction Also called citric acid cycle or the Krebs cycle (after its discoverer, Hans Krebs). TCA cycle is a series of reactions catalyzed by different enzymes in which acetyl CoA is oxidized into CO2, H2O and energy. It occurs in the mitochondrial matrix aerobically. The enzymes involved in the TCA cycle are present in the mitochondrial matrix either free or attached to the inner surface of the mitochondrial membrane.

Continue.. 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. The function of the citric acid cycle is the harvesting of high-energy electrons from carbon fuels.

Continue.. The citric acid cycle itself neither generates a large amount of ATP nor includes oxygen as a reactant. Instead, the citric acid cycle removes electrons from acetyl CoA and uses these electrons to form NADH and FADH2. The citric acid cycle includes a series of oxidation-reduction reactions that result in the oxidation of an acetyl group to two molecules of carbon dioxide. The citric acid cycle oxidizes two-carbon units, producing two molecules of CO2, one molecule of GTP, and high-energy electrons in the form of NADH and FADH2.

The amphibolic nature of TCA cycle The citric acid cycle is the gateway to the aerobic metabolism of any molecule that can be transformed into an acetyl group. The cycle is also an important source of precursors, not only for the storage forms of fuels, but also for the building blocks of many other molecules such as amino acids, nucleotide bases, cholesterol, and porphyrin. This pathway is utilized for both catabolic reactions to generate energy & anabolic reactions to generate metabolic intermediates for biosynthesis.

Metabolic pathway In oxidative phosphorylation, electrons released in the reoxidation of NADH and FADH2 flow through a series of membrane proteins to generate a proton gradient across the membrane. The citric acid cycle, in conjunction with oxidative phosphorylation, provides the vast majority of energy used by aerobic cells in human beings, greater than 95%. In TCA, the removal of high-energy electrons from carbon fuels. These electrons reduce O2 to generate a proton gradient . Which is used to synthesize ATP .

The TCA Cycle Has Eight Steps

Continue.. Step 1: Formation of Citrate - An irreversible reaction catalyzed by citrate synthase. -Inhibited by: ATP , NADH, Citrate. Step 2: Formation of Isocitrate -A reversible reaction catalyzed by aconitase . Step 3: Oxidative decarboxylation of isocitrate -The enzyme isocitrate dehydrogenase catalyzes the irreversible oxidative decarboxylation of isocitrate to form α-ketoglutarate and CO2. -Stimulated by: isocitrate, NAD+, Mn2+, ADP, Ca2+. -Inhibited by: NADH and ATP.

Continue.. Step 4: Oxidative decarboxylation of α-ketoglutarate -In this irreversible reaction, α-ketoglutarate is converted to succinyl-CoA and CO2 by the action of the α-ketoglutarate dehydrogenase complex . -α-ketoglutarate dehydrogenase complex closely resembles the PDH complex in both structure and function. -NAD+ serves as electron acceptor and CoA as the carrier of the succinyl group. - Inhibited by: NADH, ATP, Succinyl-CoA - Stimulated by: Ca2+

Continue. Step 5: Conversion of succinyl-CoA to succinate -Reversible reaction catayzed by succinyl-CoA synthetase (succinate thiokinase) -Results in the formation of GTP and CoA-SH -Nucleoside diphosphate kinase interconverts GTP and ATP by a readily reversible phosphoryl transfer reaction: GTP + ADP GDP + ATP

Continue.. Step 6: Oxidation of Succinate to Fumarate. Succinate is oxidized to fumarate by the flavoprotein succinate dehydrogenase Only TCA cycle enzyme contained within the mitochondrial membrane. Results in the formation of FADH2

Continue.. Step 7: Hydration of fumarate to malate -The reversible hydration of fumarate to L-malate is catalyzed by fumarase . Step 8: Oxidation of malate to oxaloacetate -In the last reaction of the citric acid cycle, NAD-linked L- malate dehydrogenase catalyzes the oxidation of L-malate to oxaloacetate.

Enzyme Control of the TCA Cycle

Inhibitors of TCA Cycle Fluoroacetyl CoA: -It inhibits aconitase enzyme -It combines with oxaloacetate giving rise to fluorocitrate . Malonic acid: -Inhibits succinate dehydrogenase (competitive inhibition) Arsenate and Mercury : -Inhibit Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexs. - By reacting with sulphydral group of lipoic acid leading to accumulation of pyruvic lactic acid and α- ketoglutarate.

Products of Kreb’s Cycle 2 CO2 3 NADH 1 ATP Per 1 Acetyl CoA (double for 1 glucose) 1 FADH2 ATP Yield: Each NADH yields 3 ATP Each FADH2 yields 2 ATP

Summary of total energy yield of complete oxidation of 1 glucose molecule Step Coenzyme Yield ATP Yield Source of ATP Glycolysis –Stage 1 - 2 Phosphorylation of glucose and fructose uses 2 ATP Glycolysis –Stage 2 4 Substrate level phosphorylation 2 NADH 6 Oxidative phosphorylation Pyruvate metabolism TCA cycle 2 6 NADH 18 2 FADH2 Total Yield 38 ATP