Regulation of Pyruvate Kinase and Glycolytic/Gluconeogenic Pathways

Regulation of the Pyruvate Kinase Enzyme

• Role in Glycolysis: Pyruvate kinase is the enzyme active in the final step of the payoff phase of glycolysis.

• Reaction Catalyzed: It facilitates the conversion of phosphoenolpyruvate into pyruvate.

• Energy Production: This specific enzymatic step results in the production of $ATP$.

• Isozymes Invertebrates: There are at least three isozymes of pyruvate kinase found in vertebrates. These isozymes are distinguished by:

  • Their distribution across different tissues.

  • Their specific responses to various modulators.

Allosteric Regulation and Molecular Inhibitors

• Signals of Abundant Energy: High concentrations of specific molecules signify that the cell has sufficient energy, leading to the allosteric inhibition of all pyruvate kinase isozymes.

• Primary Allosteric Inhibitors:

  • $ATP$: High levels indicate high energy charge.

  • $Acetyl\text{-}CoA$ (Acetyl coenzyme A): A product of fatty acid breakdown and a precursor for the citric acid cycle.

  • Long-chain fatty acids.

• Alanine Inhibition: Alanine is another allosteric inhibitor of pyruvate kinase.

  • Synthesis: Alanine can be synthesized directly from pyruvate through the process of transamination.

  • Mechanism: High cellular concentrations of alanine signal that the metabolic building blocks are abundant, thereby slowing down glycolysis by inhibiting the enzyme.

Tissue-Specific Isozymes: Liver vs. Muscle

• Pyruvate Kinase L (L Form): This isozyme is located in the liver.

• Pyruvate Kinase M (M Form): This isozyme is located in the muscles.

• Covalent Modification Difference: The L form (liver) is subject to regulation by phosphorylation, whereas the M form (muscle) is not subject to this type of regulation.

Hormonal Regulation of the Liver Isozyme (PK-L)

• Response to Low Blood Glucose:

  • Low blood glucose levels trigger the pancreas to release the hormone glucagon.

  • Glucagon activates cyclic $AMP$ ($cAMP$).

  • $cAMP$ activates the enzyme Protein Kinase A ($PKA$), also known as cyclic $AMP$ dependent protein kinase A.

• Phosphorylation Mechanism: $PKA$ adds a phosphate group to the liver pyruvate kinase ($PK\text{-}L$).

• Inactivation: The addition of the phosphate group renders the liver pyruvate kinase inactive.

• Metabolic Consequence:

  • Inactivation of pyruvate kinase slows down the process of glycolysis in the liver.

  • This ensures that the liver does not consume the limited glucose available, leaving it for export to critical organs like the brain.

  • Simultaneously, the demand for energy and the slowing of glycolysis stimulates or activates the process of gluconeogenesis to produce new glucose.

• Reactivation via Phosphatase:

  • When glucagon levels drop, an enzyme called protein phosphatase ($PP$) is utilized.

  • $PP$ dephosphorylates the pyruvate kinase (removes the phosphate group).

  • Removal of the phosphate group reactivates the enzyme, stimulating glycolysis.

Hormonal Regulation in Muscle Tissue

• Response to Epinephrine: In muscle tissue, increased concentrations of cyclic $AMP$ ($cAMP$) occur in response to epinephrine (the "fight or flight" hormone).

• Differentiation from Liver: Unlike the liver, increased $cAMP$ in the muscle activates glycogen breakdown and glycolysis to provide immediate fuel.

• Lack of Covalent Regulation: Pyruvate kinase in the muscle ($M$ form) is not phosphorylated by $cAMP$ dependent protein kinase; it lacks the phosphorylation-based regulation seen in the liver.

Transcriptional Regulation and CHREBP

• Concept of Transcriptional Regulation: This involves changing the total number of enzyme molecules synthesized in the cell, balancing the synthesis and breakdown of these molecules. This is distinct from fast, reversible allosteric/covalent regulation.

• CHREBP (Carbohydrate Response Element Binding Protein): This is a "Mark four" transcriptional regulator.

  • Tissue Expression: It is expressed in the liver, kidney, and adipose tissue.

  • Function: It regulates the expression of enzymes necessary for carbohydrate and fatty acid synthesis.

• Role of Xylulose 5-Phosphate:

  • In the cytosol, Xylulose 5-phosphate activates Protein Phosphatase 2A ($PP2A$).

  • $PP2A$ removes a phosphate group from the phosphorylated CHREBP (which initially has two phosphate groups).

  • Partial dephosphorylation allows the transcription factor to enter the nucleus.

  • Inside the nucleus, another instance of $PP2A$ (also stimulated by Xylulose 5-phosphate) removes the second phosphate group.

• Gene Activation:

  • The fully dephosphorylated CHREBP joins with a protein called $MLX$.

  • This complex turns on the transcription for genes encoding enzymes such as Pyruvate Kinase, Fatty Acid Synthase, and $Acetyl\text{-}CoA$ carboxylase.

• Summary of Control: Xylulose 5-phosphate control over phosphatase activity allows the cell to turn on gene expression for enzymes crucial to glycolysis and lipid synthesis.

Principles of Reciprocal Regulation

• Reciprocal Inhibition: Glycolysis and gluconeogenesis are reciprocally regulated. At any given time, the cell ensures only one process dominates to avoid a futile cycle.

• Hexokinase/Glucokinase:

  • Hexokinase (formerly known as glucokinase in the liver) is the predominant liver form.

  • Hexokinases I, II, and III are inhibited by glucose-6-phosphate, but the liver form is not.

• Phosphofructokinase-1 ($PFK\text{-}1$) and Fructose Bisphosphatase-1 ($FBPase\text{-}1$):

  • Critical glycolytic enzyme $PFK\text{-}1$ is allosterically inhibited by high concentrations of $ATP$.

  • Reciprocally, high concentrations of $ATP$ and low concentrations of $AMP$ inhibit $FBPase\text{-}1$—a key enzyme in gluconeogenesis (Note: transcript indicates high $ATP$ inhibits $FBPase\text{-}1$ in this context).

  • High $ATP$ slows glycolysis but can speed up gluconeogenesis based on cellular energy demands.

• Fructose 2,6-bisphosphate ($F26BP$): This is a vital intermediate for residual allosteric control. It has opposite effects on $PFK\text{-}1$ and $FBPase\text{-}1$.

  • Formation is indirectly triggered by insulin and inhibited by epinephrine or glucagon.

• Xylulose 5-Phosphate Activity: It activates $PP2A$, which tips the metabolic balance toward glucose uptake, glycogen synthesis, and lipid synthesis in the liver.

• Acetyl-CoA in Mitochondria: Fatty acid breakdown in liver mitochondria yields $Acetyl\text{-}CoA$, which activates pyruvate carboxylase, thereby favoring gluconeogenesis.

Questions & Discussion

• Scenario Problem: In a cell, the concentration of $ATP$ is low.

• Prompt for Consideration: Predict what would happen to the following:

  1. The activity of Fructose 1,6-bisphosphatase 1 ($FBPase\text{-}1$).

  2. The rate of glycolysis.

  3. The rate of gluconeogenesis.

• Context: This problem is designed for further thought and discussion in upcoming workshops regarding how low energy charge shifts metabolic pathways.