Dr. Abir Alghanouchi Biochemistry department Sciences college

Dr. Abir Alghanouchi Biochemistry department Sciences college

Under aerobic conditions, pyruvate(the product of glycolysis) passes by special pyruvatetransporter into mitochondria which proceeds as follows: 1. Oxidative decarboxylation of pyruvate into acetyl CoA. 2. Acetyl CoA is then oxidized completely to CO 2, H 2 O through Krebs' cycle G first stage cytosol glycolytic pathway Pyr Mitochodria second stage Pyr CH 3 CO~SCoA third stage CO2 + H 2 O+ATP TAC

COO - C CH 3 pyruvate NAD + NADH + H + O + HSCoA H 3 C C~SCoA + CO 2 Pyruvate dehydrogenase complex Irreversible reaction catalyzed by a multi enzyme complex associated within the inner mitochondrial membrane known as Pyruvate dehydrogenase complex O Acetyl CoA

Pyruvate dehydrogenase complex This enzyme complex contains 3 subunits, which catalyze the reaction in 3 steps: E 1 pyruvatedehydrogenase Es E 2 dihydrolipoyltransacetylase E 3 dihydrolipoyldehydrogenase HSCoA NAD +

Pyruvate dehydrogenase complex This enzyme needs 5 coenzymes (all are vitamin B complex derivatives) Thiamine pyrophosphate, TPP (VB 1 ) HSCoA(pantothenicacid) cofactors lipoic Acid NAD + FAD (VB 2 ) HSCoA NAD +

Regulation of Pyruvate Dehydrogenase Irreversible reaction must be tightly controlled-- three ways Allosteric Inhibition Inhibited by products: acetyl-coaand NADH Inhibited by high ATP Allosteric activation by AMP Ratio ATP/AMP important

Covalent modification (hormonal regulation): Through Phosphorylation/dephosphorylation of E1 subunit PDHexists in two forms: Phosphorylated(inactive): Protein kinaseenzyme converts active into inactive enzyme Dephosphorylated(active): Phosphataseenzyme converts inactive into active NB: In vitro inhibition of PDH: Arsenic Mercury

E and product accumulation: 1 2 allosteric inhibitors: ATP, acetyl CoA, NADH, FA Low levels E: allosteric activators: AMP, CoA, NAD +,Ca 2+ Pyruvate dehydrogenase (Active dephosphorylated (active form) form) Pi pyruvate dehydrogenase phosphatase H 2 O ATP pyruvate dehydrogenase kinase ADP 3 Ca 2+,insulin pyruvate dehydrogenase P (inactive form) (Inactive phosphorylated form) acetyl CoA, NADH ADP, NAD + Regulation of E1 by covalent modification through phosphorylation

Acetyl CoAis an important molecule in metabolism used in many biochemical reactions Acetyl CoA functions as: 1. input to Krebs Cycle, where the acetate moiety is further degraded to CO 2 2. donor of acetate for synthesis of FA, ketone bodies, & cholesterol citric acid cycle GLUCOSE glycolysis PYRUVATE Acetyl CoA pyruvate dehydrogenase lipogenesis β-oxidation Fatty acids (Cytoplasm) CO 2 In mammals, acetyl CoA is essential to the balance between CHO and fat metabolism ketogenesis (liver only) Ketone bodies ketone oxidation Cholesterol cholesterol synthesis steroid hormones (endocrine glands) Figure: Metabolic sources and fates acetyl CoA

gluconeogenesis alanine aminotransferase Alanine GLUCOSE glycolysis PYRUVATE pyruvate carboxylase lactate dehydrogenase pyruvate dehydrogenase Lactate Oxaloacetate citric acid cycle Acetyl CoA lipogenesis β-oxidation Fatty acids (Cytoplasm) CO 2 ketogenesis (liver only) ketone oxidation cholesterol synthesis Ketone bodies Cholesterol steroid hormones (endocrine glands) Figure: Metabolic sources and fates of pyruvate and acetyl CoA

Kreb'scycle Also known as Citric Acid Cycle (CAC) Or Tricarboxylic Acid Cycle (TCA) Or Catabolism of Acetyl CoA (CAC)

Cytosol Mitochondrion Glycolysis 2 Glucose Pyruvic acid 2 Krebs Acetyl- Cycle CoA Electron Transport Maximum per glucose: by direct synthesis by direct synthesis by ATP synthase

Definition: TCA is a series of enzyme-catalyzed chemical reactions in which acetyl CoA is oxidized intoco 2,H 2 Oandenergy. Location: Occurs in the matrix of the mitochondrion = aerobically

Steps: othe enzymes of TCA are present in the mitochondrial matrix either free or attached to the inner surface of the mitochondrial membrane. othe cycle is started by acetyl CoA(2C)and oxaloacetate(4 C) to form citrate (6C). It ends by oxaloacetate(4c). othe difference between the starting compound (6C)and the ending compound (4C)is 2 carbons that are removed in the form of 2 CO 2. These 2 carbons are derived from acetyl CoA. For this reason acetyl CoAis completely catabolized in TCA and never gives glucose.

The cycle begins with the condensation of acetyl-coa and oxaloacetate to form citrate Non-equilibrium reaction catalyzed by citrate synthase Inhibited by: ATP NADH Citrate - competitive inhibitor of oxaloacetate

Aconitase then catalyzes the interconversion of citrate and isocitrate via dehydration and hydration Equilibrium reactions Results in interchange of H and OH

Isocitrate is then converted to α-ketoglutarate via oxidative decarboxylation, producing CO 2 Isocitrate dehydrogenated and decarboxylated to give α- ketoglutarate Non-equilibrium reaction catalyzed by isocitrate dehydrogenase + NAD

Isocitrate is then converted to α-ketoglutarate via oxidative decarboxylation, producing CO 2 Results in formation of: o NADH + H + o CO 2 Stimulated by isocitrate, NAD +, Mg 2+, ADP, Ca 2+ Inhibited by NADH and ATP + NAD

The α-ketoglutarate is then converted to succinyl-coa via another oxidative decarboxylation, producing the second CO 2 Series of reactions result in decarboxylation, dehydrogenation and incorporation of CoASH Non-equilibriumreactions catalyzed by α-ketoglutarate dehydrogenase complex Stimulated by Ca 2+ Inhibited by NADH, ATP, Succinyl CoA TPP lipoate FAD

Succinyl CoA is the converted to succinate, accompanied by the formation of a GTP (or ATP) Equilibrium reaction catalyzed by succinate thiokinase Results in formation of GTP and CoA-SH Nucleoside diphosphatekinaseinterconvertsgtp and ATP by a readily reversible phosphoryltransfer reaction: GTP + ADP GDP + ATP

Succinate is then converted to fumarate by dehydrogenation Succinate dehydrogenated to form fumarate Equilibrium reaction catalyzed by succinate dehydrogenase Only Krebs enzyme contained within inner mitochondrial membrane Results in formation of FADH 2

Fumarate is then converted to malate via hydration Equilibrium reaction catalyzed by fumarase Results in redistribution of energy within molecule so next step can remove hydrogen

Malate dehydrogenated to form oxaloacetate Equilibrium reaction catalyzed by malate dehydrogenase Results in formation of NADH + H +

Glucose glycolysis ------ PDH Pyruvate fatty acids, ketone bodies Acetyl CoA CoA Figure: Reactions of the citric acid cycle NADH ------ NAD + Malate Oxaloacetate -------- Citrate cis Aconitate Isocitrate α-ketoglutarate NAD ------ + NADH, ------ CO 2 CoA, NAD ------ + Fumarate Succinyl ------CoA NADH, ------ CO 2 FADH ------ 2 FAD Succinate GTP GDP ------ ATP ADP

Products of Krebs Cycle 2 CO 2 3 NADH 1 ATP 1 FADH 2 ATP Yield Each NADH energizes 3 ATP Each FADH 2 energizes2 ATP Double this list for each glucose

Step Glycolysis preparatory phase Glycolysis pay-off phase Oxidative decarboxylation of pyruvate Coenzyme Yield ATP Yield Source of ATP -2 Phosphorylation of glucose and fructose uses 2 ATP 4 Substrate level phosphorylation 2 NADH 6 Oxidative Phosphorylation 2 NADH 6 Oxidative Phosphorylation Krebs Cycle 2 Substrate level phosphorylation Total Yield 38 ATP 6 NADH 18 Oxidative Phosphorylation 2 FADH 2 4 Oxidative Phosphorylation Complete oxidation of one glucose molecule to CO2 and oxidation of all the reduced coenzymes

The amphibolicnature of Citric acid cycle By transamination, oxaloacetate is converted to aspartate By transamination α-ketoglutarate is converted to glutamate This pathway is utilized for the both catabolic reactions to generate energy as well as for anabolic reactions to generate metabolic intermediates for biosynthesis

What are the key regulated enzymes in citrate cycle?

FluoroacetylCoA: it combines with oxaloacetategiving rise to fluorocitrate which inhibits aconitase enzyme Malonicacid: inhibits succinatedehydrogenase (competitive inhibition) Arsenate and Mercury : inhibit Pyruvate dehydrogenase and α-ketoglutaratedehydrogenase complex by reacting with sulphydralgroup of lipoicacid leading to accumulation of pyruvic lactic acid and α- ketoglutarate with acidosis

The Glyoxylate Cycle Another Process Involving Glycolytic Enzymes and Metabolites

Anabolic metabolic pathway occurring in plants, and several microorganisms, not animals. Occurs in glyoxysome The enzymes common to the TCA cycle and the glyoxysomesare isoenzymes, one specific to mitochondria and the other to glyoxysomes. The glyoxylate cycle allows plants to use acetyl-coa derived from β-oxidation of fatty acids for carbohydrate synthesis Animals can not do this! Acetyl-CoAis totally oxidized to CO 2

The Glyoxylate Cycle: Demonstration of Connections to the Citric Acid Cycle 1. Citrate converted to isocitrate, then it is cleaved by isocitrate lyase forming succinate and glyoxylate 2. Glyoxylate condenses with 2 sd acetyl CoA to yield malate by malate synthase 3. Malate is then oxidized to oxloacetate which will condense with another molecule of Acetyl CoA to start another turn 4. Or enters the cytosol and oxidized to oxaloacetate (precursor of glucose via gluconeogensis)

The Glyoxylate Cycle: Demonstration of Connections to the Citric Acid Cycle 5. Succinate returns to mitochondria, where it re enters the TCA cycle and is transformed to oxaloacetate, which can again be exported (via aspartate) to the glyoxysome Each turn of this cycle consumes 2 molecules of Acetyl CoAcycle and produce 1 molecule of succinate Note that the decarboxylation reactions of the citric acid cycle are bypassed

Some bateria, including E Coli, have the full complement of enzymes for the glyoxylate and TCA cycles in the cytosol E coli can therefore grow with acetate as its sole source of carbon and energy sole source of carbon and energy

----------- Pyruvate is converted to acetyl-coaby the action of pyruvate ---------------------- dehydrogenase complex, a huge enzyme complex. Acetyl-CoAis converted to 2 CO 2 via the eight-step citric ----------- acid cycle, generating three NADH, one FADH 2, and one ATP (by substrate-level phophorylation). Intermediates of citric acid cycle are also used as biosynthetic ----------- precursorsfor --------- many other biomolecules, including fatty acids, steroids, amino acids, heme, pyrimidines, and glucose. Oxaloacetatecan get replenished from pyruvate, via a carboxylation reaction catalyzed by pyruvate carboxylase. -----------------

The activity of pyruvatedehydrogenase complex is regulated by --------- allosteric effectors and reversible phosphorylations. -------------- Net conversion of fatty acids to glucose can occur in germinating seeds, some invertebrates and some bacteria via the glycoxylate ---------- ----- cycle, which shares three steps with the citric acid cycle but bypasses the two ---------------------- decarboxylation steps, converting two molecules of acetyl- CoA to one succinate.