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Pyruvate Dehydrogenase and The TCA Cycle

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Outline
Cellular Logic of Pathways
*All catabolic pathways lead ultimately to Pyruvate and Acetyl CoA
Major Source of Energy for ATP Synthesis in Oxygen-Dependent Tissues
*Oxidation of Acetyl CoA in the Tricarboxylic Acid Cycle and the accompanying Oxidation of Reduced Coenzyme Products
Final Product of Pyruvate Dehydrogenase (PDH)
*Acetyl Coenzyme A (Acetyl CoA)
-Also produced by fatty acid metabolism & some amino acid conversions
Oxidation of Pyruvate
*Occurs in Mitochondrial Matrix via PDH & the Krebs Cycle
*Products = Acetyl-CoA & Carbon Dioxide
Pyruvate Dehydrogenase (PDH) - Products of Pyruvate Oxidation
*Products of oxidation of Pyruvate
-Acetyl CoA and Carbon Dioxide
Pyruvate Dehydrogenase (PDH) Structure
*Multienzyme complex
-Functionally related enzymes that are physically associated with each other
Pyruvate Dehydrogenase (PDH) Coenzyme-cosubstrates (2)
1) NAD
2) CoA
Pyruvate Dehydrogenase (PDH) Catalytic Coenzyme Prosthetic Groups (3)
1) Thiamine Pyrophosphate (E1)
2) FAD (E3)
3) Lipoic Acid (E2)
Pyruvate Dehydrogenase (PDH) Enzymes (3)
1) E1
2) E2
3) E3
Pyruvate Dehydrogenase (PDH) Regulatory Enzymes (2)
1) Kinase
2) Phosphatase
Pyruvate Dehydrogenase (PDH) Visual Interpretation
Pyruvate ---> Acetyl-CoA By PDH (Step 1)
1) Decarboxylation by E1 containing Thiamine Pyrophosphate
*The keto carbon of pyruvate reacts with the carbanion of TPP on E1 to yield an addition compound. The electron-pulling positively charged nitrogen of the thiazole ring promotes loss of CO2. What remains is hydroxyethyl-TPP.
Pyruvate ---> Acetyl-CoA By PDH (Step 2)
2) Oxidation of product by E2 utilizing Lipoamide & subsequent transfer of Acetyl group to CoA
*The hydroxyethyl carbanion on TPP of E1 reacts with the disulfide of lipoamide on E2. What was the keto carbon of pyruvate is oxidized to a carboxylic acid, as the disulfide of lipoamide is reduced to a dithiol. The acetate formed by oxidation of the hydroxyethyl moiety is linked to one of the thiols of the reduced lipoamide as a thioester (~).
*The acetate is transferred from the thiol of lipoamide to the thiol of coenzyme A, yielding acetyl CoA.
*The reduced lipoamide swings over to the E3 active site. Dihydrolipoamide is reoxidized to the disulfide, as 2 e- + 2 H+ are transferred to a disulfide on E3 (disulfide interchange).
Pyruvate ---> Acetyl-CoA By PDH (Step 3)
3) Catalyzed by E3, Reoxidation of Lipoamide by Flavin adenine dinucleotide (FAD) & subsequent reduction of nicotinamide adenine dinucleotide (NAD)
*Acetyl intermediate covalently bound to enzyme complex by lipoamide
*The dithiol on E3 is reoxidized as 2 e- + 2 H+ are transferred to FAD. The resulting FADH2 is reoxidized by electron transfer to NAD+, to yield NADH + H+.
Lipoyl Lysine side chain of Dihydrolipoyl Transacetylase (E2)
*Serves as swinging arm to transfer electrons from Pyruvate Decarboxylase (E1) ---> Dihydrolipoyl Dehydrogenase (E3)
Control of Pyruvate Dehydrogenase
*Covalent Inhibition (Enzymic phosphorylation - E1)
*Insulin Upregulates
Covalent Regulation of PDH
*Depends on a specific protein Kinase and Phosphatase Pair
*PDH Kinase & Phosphatase are physically associated with the PDH supramolecular complex
PDH Kinase
*Stimulated by ATP, NADH, and Acetyl CoA
*Inhibited by Pyruvate
PDH Phosphatase
*Activated by Calcium
-Calcium is released by sarcoplasmic reticulum of muscles upon utilization of ATP
-PDH activity upregulated in response to deprevation of energy source
The Citric Acid Cycle - (Krebs Cycle) (TCA Cycle)
*Acetyl CoA produced by Pyruvate Dehydrogenase (PDH) as well as by Beta-oxidation of fatty acids utilized in the Tricarboxylic Acid Cycle
Mnemonic for Molecules ("Can Adam & I Keep Selling Sex For Money, Officer?")
-Citrate
-cis-Aconitate
-Isocitrate
-alpha Ketogluterate
-Succinyl CoA
-Succinate
-Fumerate
-Malate
-Oxaloacetate
Mnemonic for Enzymes ("So At Another Dance Devon Sipped Down Five Drinks")
*Remember molecule that precedes enzyme
- citrate Synthase
- Aconitase
- Aconitase
- isocitrate Dehydrogenase
- alpha-ketoglutarate Dehydrogenase
- Succinyl-CoA Synthetase
- Succinate Dehydrogenase
- Fumarate
- malate Dehydrogenase
Primary Function of TCA Cycle
*Oxidation of acetyl CoA to 2 Carbon Dioxide
-Results in generation of ATP (Substrate-level Phosphorylation & Oxidative Phosphorylation)
Location within Mitochondria
*Mitochondrial Matrix
Cyclic Nature
*Implies a catalytic role for axolacetate and other intermediates
*NOT POSSIBLE to generate intermediates from acetyl CoA alone
Enzymes of TCA Cycle
*EXCEPTION succinate Dehydrogenase (Embedded in inner mitochondrial membrane)
*Soluble and are found in the Matrix
*Intermediates "free" to interact with other enzyme pathways (If this occurs...necessary to replace the intermediates from other sources)
The Initiating Reaction
*Catalyzed by Citrate Synthase
*Irreversible ---> Hydrolysis of the thioester bond of acetyl CoA
*Example of Enzyme-Bound Intermediate
Aconitase
*Derives its name from cis-aconitate intermediate
*Functions as a dehydrase/hydrase (Removes Water and then Adds it Back)
-Results in a different location of the -OH group
Oxidation-Reductions
*Four reactions are oxidation-reductions
-3 linked to NAD+ reduction
-1 (Succinate Dehydrogenase) linked to FAD reduction
*Products, NADH and FADH2 are specific intermediates ---> Generation of ATP (Oxidative Phosphorylation)
Production of Carbon Dioxide
*Isocitrate dehydrogenase & alpha-ketoglutarate dehydrogenase
-Result in production of Carbon Dioxide
***E3 is common to both PDH and alpha-ketoglutarate dehydrogenase (These two reactions along with pyruvate oxidation account for major part of Carbon Dioxide produced and exhaled)
GTP Generation
*GTP generated from Succinyl CoA
-Example of substrate level phosphorylation
*Exergonic reaction (large -ΔG) catalyzed by the α-ketoglutarate dehydrogenase and the synthesis of the high energy phosphate bond in GTP (large +ΔG) catalyzed by the succinyl thiokinase are coupled through the succinyl-CoA derived from α-ketoglutarate.
Succinyl Thiokinase (Succinyl CoA Synthetase)
*Hydrolysis of succinyl CoA is COUPLED to synthesis of GTP
-Example of energy coupling where common intermediate can only be found bound to enzyme
*Thioester Bond = High ENERGY
The ΔG Of Pyruvate Oxidation
TCA Cycle overall is EXERGONIC and IRREVERSIBLE operates under AEROBIC Conditions
Coupled Reactions
*Malate oxidation by NAD+ (ΔG = +7)
-However Endergonic reactions are COUPLED with Exergonic reactions
*TOTAL ΔG = NEGATIVE
Irreversible Steps (3)
1) Citrate synthase
2) Isocitrate dehydrogenase
3) Alpha-keto-glutarate dehydrogenase reactions
*Prevents Cycle from Reversing Direction
*May be the result of HIGHLY -ΔG or Hydrolytic Bond Cleavage
* In Isocitrate dehydrogenase reaction, product (NADH) is inhibitory & Carbon Dioxide reacts with Water
Regulation of TCA Cycle
*Primary sites of regulation are at the IRREVERSIBLE SITES
Citrate Synthase
*Competitively inhibited by Citrate
Isocitrate Dehydrogenase
***Usually considered primary site of control
*Inhibited allosterically by NADH & ATP
*Positive Effector - ADP
*Activator - Calcium
TCA Cycle as a Source of Metabolic Substrates
Replenishing of TCA Cycle Intermediates