Comprehensive Biochemistry: Glycolysis, Gluconeogenesis, Beta-Oxidation, and the Citric Acid Cycle

Glycolysis and Gluconeogenesis: The Cori Cycle and Pathway Overviews

Functional Sites and Distinctions
    - Liver: The primary site for gluconeogenesis (represented in grey/gluconeogenesis descriptions).
    - Muscle: Primarily undergoes glycolysis (represented in black) and muscle fermentation under anaerobic conditions.
    - Cori Cycle: Involves the movement of lactate from the muscle to the liver for gluconeogenesis and the return of glucose to the muscle.
Glucose Transporters
    - GLUT4: A glucose transporter found in adipose and muscle tissues that facilitates glucose uptake, typically regulated by insulin.
    - GLUT2: A glucose transporter found in the liver that facilitates glucose transport across the membrane.
    - MCT4: Transports lactate produced in the muscle to the liver.
Glycolysis Enzyme Pathway and Intermediates
    - Hexokinase II: Catalyzes the first step of glycolysis: the phosphorylation of glucose to glucose-6-phosphate.
    - Phosphoglucose isomerase: Converts glucose-6-phosphate into fructose-6-phosphate.
    - Phosphoglycerate mutase: Regulated by AMP levels; it forms an active tetramer by binding AMP at an allosteric site to produce fructose-1,6-bisphosphate.
    - Aldolase: Catalyzes the cleavage of fructose-1,6-bisphosphate into two three-carbon molecules: glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP).
    - Triose-phosphate Isomerase: Interconverts DHAP and G3P.
    - GAPDH (Glyceraldehyde-3-phosphate dehydrogenase): The redox enzyme of glycolysis. It catalyzes the conversion of G3P into 1,3-bisphosphoglycerate. It requires $NAD^+$ as a crucial limiting substrate.
    - Phosphoglycerate kinase: Converts 1,3-bisphosphoglycerate to 3-phosphoglycerate, producing ATP from ADP via substrate-level phosphorylation.
    - Phosphoglycerate mutase: Converts 3-phosphoglycerate to 2-phosphoglycerate.
    - Enolase: Converts 2-phosphoglycerate into phosphoenolpyruvate (PEP) with the release of $H_2O$ .
    - Pyruvate kinase: Catalyzes the final step of glycolysis, converting PEP to pyruvate and producing ATP.
    - LDH (Lactate Dehydrogenase): In the absence of oxygen (anaerobic conditions), LDH reduces pyruvate to lactate to regenerate $NAD^+$ from $NADH$ .
Gluconeogenesis Enzyme Pathway and Intermediates
    - Pyruvate Carboxylase: Catalyzes the carboxylation of pyruvate to oxaloacetate (OAA) within the mitochondrial matrix. This requires $ATP$ and $CO_2$ .
    - PEPCK (Phosphoenolpyruvate carboxykinase): Converts OAA to phosphoenolpyruvate (PEP), utilizing $GTP$ and releasing $CO_2$ .
    - Fructose-1,6-bisphosphatase: Converts fructose-1,6-bisphosphate back into fructose-6-phosphate, bypassing the irreversible PFK1 step.
    - Glucose-6-phosphatase: The final enzyme in the liver that converts glucose-6-phosphate into free glucose, allowing its release into the bloodstream to maintain blood sugar levels.
Energy Yield and Considerations
- Glycolysis Energy: Produces a total of $4$ ATPs; however, since $2$ ATPs are consumed in the early "investment" phase, the net gain is $2$ ATPs per glucose molecule. $NAD^+$ is reduced to $NADH$ .
- Gluconeogenesis Energy: This is an anabolic process that consumes energy. It requires a total of $6$ ATP equivalents (including $GTP$ ) to generate one molecule of glucose.

Beta Oxidation and Ketogenesis

The Carnitine Shuttle and Fatty Acid Entry
    1. Acyl CoA synthetase: Modifies fatty acids entering the cytoplasm to allow them to be used in beta-oxidation.
    2. Fatty acid translocase: Facilitated by Rab8a, which guides the assembly and movement of vesicles containing this translocase when AMP levels are high.
    3. Malonyl CoA: The substrate for fatty acid synthase; it acts as an inhibitor of the movement of acyl-chains into the mitochondrial matrix.
    4. Carnitine translocase: Located in the mitochondrial inner membrane; it transfers acyl-chains to the matrix where they are reattached to CoAs.
Stages of Beta-Oxidation
    5. The first step involves the two-step oxidation of acyl-CoAs by acyl CoA DH (dehydrogenase) in the matrix, which is linked to the reduction of FAD.
    6. 2-enoyl-CoA: The oxidized product of the acyl-CoA DH reaction.
    7. 3-hydroxyacyl-CoA: Produced when water is reacted with 2-enoyl-CoA to prepare for the second oxidation.
    8. 3-ketoacyl-CoA: The product of 3-hydroxyacyl dehydrogenase.
    9. NAD+ Reduction: The oxidation of 3-hydroxyacyl-CoA is coupled to the reduction of $NAD^+$ to $NADH$ .
    10. Thiolase: Reacts 3-ketoacyl-CoA with CoA at the beta-keto group.
    11. Palmitoyl-CoA and Acetyl-CoA: Thiolase produces these at the end of one round of beta-oxidation of stearoyl-CoA.
Ketogenesis (Low Pyruvate Conditions)
    12. Acetoacetyl-CoA: Formed when pyruvate is low within the matrix during beta-oxidation; thiolase/thiolate converts acetyl-CoA into this intermediate.
    13. HMG-CoA: The 6-carbon-CoA intermediate produced during the ketogenesis pathway.
    14. Acetone: The three-carbon waste product of ketogenesis.
    15. Beta-hydroxybutyrate: The enzymatically reduced product of ketogenesis. This compound is utilized by the Central Nervous System (CNS) to survive periods of hypoglycemia.

NADH Shuttling Mechanics

Cytoplasmic Glycolysis and Regulation
    - Adenylate kinase: An enzyme that produces AMP as a byproduct during the conversion of ADP to ATP; AMP acts as a signal that ATP levels are low.
    - PFK1 Activation: AMP activates PFK1 to form the active tetramer.
    - GAPDH Substrate: $NAD^+$ is the crucial limiting substrate that sets the overall rate of glycolysis when ATP levels are low.
Path A: Malate-Aspartate Shuttle (Heart Muscle and Liver)
    - NADH generated by GAPDH is used to reduce OAA (oxaloacetate) to malate, catalyzed by cytoplasmic malate dehydrogenase (MDH).
    - Malate is transported into the mitochondrial matrix in exchange for the export of alpha-ketoglutarate.
    - ASP+ (Aspartate Aminotransferase): Catalyzes the convertibility within the matrix of $OAA + glutamate \rightarrow aspartate + alpha-ketoglutarate$ .
    - Aspartate is then exported to the cytoplasm in exchange for glutamate entering the matrix.
Path B: Glycerol-3-Phosphate (G3P) Shuttle (White/Oxidative Muscle)
    - Glycerol-3-phosphate is produced by the reduction of DHAP.
    - During exertion, $NAD^+$ is produced by a protein integral to the mitochondrial inner membrane that oxidizes G3P.
    - Mitochondrial inner membrane G3P-dehydrogenase reduces FAD to $FADH_2$ .
Path C: Lactate Fermentation
- Occurs in the muscle; creation of $NAD^+$ is achieved by reducing Pyruvate to lactate.
Final ATP Yields
- Oxidation of malate in the matrix (via Malate-Aspartate shuttle) produces NADH, which generates approximately 2.5 ATP via oxidative phosphorylation.
- Oxidation of G3P at the inner membrane results in 1.5 ATP.

Citric Acid Cycle (CAC) Enzymology

Entry and The PDH Complex
    1. Pyruvate: The three-carbon compound that enters the mitochondrial matrix via a proton gradient across the inner membrane.
    2. Pyruvate dehydrogenase (PDH) complex: Decarboxylates pyruvate, donates a hydride to $NAD^+$ , and forms acetyl CoA.
    3. Arithmetic 3-1=2: PDH performs this arithmetic (3-carbon pyruvate loses 1 carbon to $CO_2$ to become 2-carbon Acetyl-CoA).
    4. Thioester bond: Carbons 2 and 3 of pyruvate form a high-energy thioester bond with CoA.
    5. PDH Complex: Responsible for forming that high-energy thioester bond.
Cofactors and Mechanisms
    6. Lipoamide: An elongated cofactor attached to the alpha subunits of both PDH and alpha-KGDH complexes. It undergoes reductive alkylation, dealkylation (preserving the reduced state), and oxidation to produce $FADH_2$ .
    7. Lysine: Lipoamide is covalently linked to a lysine residue via an amide bond on a subunit of the dehydrogenase complexes.
    8. Citrate synthase: Performs the arithmetic 4+2=6 ( $OAA + Acetyl-CoA \rightarrow Citrate$ ).
    9. Carboxylate: The orbital involved in the formation of a third carboxylate in citrate.
    10. Isocitrate dehydrogenase: Produces the $CO_2$ that causes the arithmetic 6-1=5.
    11. Energetics: While the formation of a keto group and reduction of $NAD^+$ are endergonic, the total \Delta G^\circ' of isocitrate dehydrogenase is -2 kcal/mol because of the simultaneous formation of $CO_2$ .
    12. Alpha-ketoglutarate dehydrogenase: Performs the arithmetic 5-1=4.
    13. Substrate-level phosphorylation: The reaction of succinyl CoA synthetase produces a high-energy phosphate bond.
    14. Nucleoside Triphosphate: The thioester bond in succinyl-CoA drives the formation of ATP or GTP through substrate-level phosphorylation.
Succinate and Malate Reactions
    15. Histidine: Succinate dehydrogenase (Complex II) draws a hydride from succinate and donates it to FAD, which is covalently linked to the active site by a histidine.
    16. Fumarase: Hydrates the oxidized product of succinate dehydrogenase (fumarate) to produce malate.
    17. Aspartate: Malate dehydrogenase (MDH) uses a histidine to remove a proton from malate's C2-OH; this action is stabilized by an aspartate residue.
    18. Arginine: The C2 oxaloacetate that forms during malate oxidation is stabilized by a protonated positively charged histidine and an active site arginine.
    19. Malate dehydrogenase: Uses $NAD^+$ as the electron acceptor to convert a 4-carbon compound (malate) into another 4-carbon compound (OAA).
    20. Lipoamide: Contains a residue providing the nucleophilic sulfur in CoA (though not technically a protein amino acid, it is a crucial cofactor component).

Entry and The PDH Complex
- Pyruvate: The three-carbon compound that enters the mitochondrial matrix via a proton gradient across the inner membrane.
- Pyruvate dehydrogenase (PDH) complex: Decarboxylates pyruvate, donates a hydride to $NAD^+$ , and forms acetyl CoA.
- Arithmetic 3-1=2: PDH performs this arithmetic (3-carbon pyruvate loses 1 carbon to $CO_2$ to become 2-carbon Acetyl-CoA).
- Thioester bond: Carbons 2 and 3 of pyruvate form a high-energy thioester bond with CoA.
- PDH Complex: Responsible for forming that high-energy thioester bond.
Cofactors and Mechanisms
- Lipoamide: An elongated cofactor attached to the alpha subunits of both PDH and alpha-KGDH complexes. It undergoes reductive alkylation, dealkylation (preserving the reduced state), and oxidation to produce $FADH_2$ .
- Lysine: Lipoamide is covalently linked to a lysine residue via an amide bond on a subunit of the dehydrogenase complexes.
- Citrate synthase: Performs the arithmetic 4+2=6 ( $OAA + Acetyl-CoA \rightarrow Citrate$ ).
- Carboxylate: The orbital involved in the formation of a third carboxylate in citrate.
- Isocitrate dehydrogenase: Produces the $CO_2$ that causes the arithmetic 6-1=5.
- Energetics: While the formation of a keto group and reduction of $NAD^+$ are endergonic, the total \Delta G^\circ' of isocitrate dehydrogenase is -2 kcal/mol because of the simultaneous formation of $CO_2$ .
- Alpha-ketoglutarate dehydrogenase: Performs the arithmetic 5-1=4.
- Substrate-level phosphorylation: The reaction of succinyl CoA synthetase produces a high-energy phosphate bond.
- Nucleoside Triphosphate: The thioester bond in succinyl-CoA drives the formation of ATP or GTP through substrate-level phosphorylation.
Succinate and Malate Reactions
- Histidine: Succinate dehydrogenase (Complex II) draws a hydride from succinate and donates it to FAD, which is covalently linked to the active site by a histidine.
- Fumarase: Hydrates the oxidized product of succinate dehydrogenase (fumarate) to produce malate.
- Aspartate: Malate dehydrogenase (MDH) uses a histidine to remove a proton from malate's C2-OH; this action is stabilized by an aspartate residue.
- Arginine: The C2 oxaloacetate that forms during malate oxidation is stabilized by a protonated positively charged histidine and an active site arginine.
- Malate dehydrogenase: Uses $NAD^+$ as the electron acceptor to convert a 4-carbon compound (malate) into another 4-carbon compound (OAA).
- Lipoamide: Contains a residue providing the nucleophilic sulfur in CoA (though not technically a protein amino acid, it is a crucial cofactor component.