Enzyme Regulation

• Metabolic pathway


  The enzymatic route that a metabolite molecule takes
• Metabolic control via enzyme regulation

  • Regulation of metabolism
  • Identifying rate-limiting steps (RLS)
  • Types of enzyme regulation
• Identifying RLS


  1. Measure Vmax for enzymes in pathway, lowest is RLS

  2. Compare Keq and mass action ratios (MAR)

  3. Test if step is at crossover point
• Slowest reaction in pathway determines overall flux through the pathway by acting as a bottleneck
• Mass action ratio (MAR)


  Ratio of products to substrates within the cell
• If reaction is rate-limiting in the cell

  MAR is usually 100-1000 times smaller than Keq
• Crossover point identifies regulatory step. If pathway flux increases, activity of enzyme controlling flux must have increased
• The need for control

  • 1) Ensure a given metabolic pathway is active when its product is needed
  • 2) Ensure competing pathways aren't simultaneously active
  • 3) Ensure co-ordinated activity in multiple related pathways
• Types of enzyme regulation

  • Intrinsic control by metabolites: Allosterism/inhibitors
  • Fast extrinsic control via hormones: Covalent modification - e.g. phosphorylation
  • Slow extrinsic control via hormones: Gene expression
• Control by metabolites

  • Product inhibition
  • Allosterism
• Metabolites controlling enzymes


  • ATP inhibiting Hexokinase
  • G-6-P inhibiting its own production
  • Fructose 2,6-bisphosphate activating PFK-1 and inhibiting F-1,6-bisphosphatase
  • ADP/AMP activating, ATP inhibiting various enzymes
• Phosphorylation

  • Conformational change leading to activation or inactivation of enzyme
  • Phosphorylation overrides allosterism
• Enzymes regulated by phosphorylation

  • Phosphorylase b (inactive) to Phosphorylase a (active)
  • Glycogen synthase
• Adrenaline/Glucagon

  Increase glycogenolysis, decrease glycogenesis
• Insulin


  Increases glycogenesis, decreases glycogenolysis
• Glycogen metabolism is regulated by phosphorylation via hormones
• Define a rate-limiting step in a metabolic pathway

  The rate-limiting step is the slowest reaction in a metabolic pathway, which determines the overall flux through the pathway by acting as a bottleneck
• How can the rate-limiting step in a pathway be determined?

  The rate-limiting step can be determined by measuring the Vmax for enzymes in the pathway (the enzyme with the lowest Vmax might be the rate-limiting step), comparing the Keq and mass action ratios (MAR), and testing if a step is at a crossover point by perturbing the system and observing changes in metabolite concentrations
• Explain the concept of a futile cycle and why it should be avoided

  A futile cycle occurs when two metabolic pathways run simultaneously in opposite directions, leading to the consumption of energy without producing useful work. It should be avoided because it wastes energy and resources
• Describe product inhibition in enzyme regulation

  Product inhibition occurs when the product of an enzyme-catalyzed reaction binds to the enzyme and decreases its activity, thus regulating the pathway by preventing the overaccumulation of the product
• Describe allosterism in enzyme regulation

  Allosterism involves the regulation of an enzyme's activity through the binding of a molecule at a site other than the active site, which induces a conformational change in the enzyme that affects its activity
• How do ATP and ADP regulate glycolysis and TCA cycle enzymes?

  ATP generally inhibits enzymes in glycolysis and the TCA cycle to prevent excess energy production, while ADP/AMP activate these enzymes to stimulate energy production when cellular energy levels are low
• Explain how hormones regulate metabolic pathways via phosphorylation and dephosphorylation

  Hormones such as insulin, adrenaline, and glucagon regulate metabolic pathways by causing the phosphorylation or dephosphorylation of key enzymes, which can activate or inhibit these enzymes and thereby control the pathway's activity
• How do hormones and metabolites regulate the expression of enzyme genes?

  Hormones can induce or repress the transcription of genes encoding metabolic enzymes, leading to changes in enzyme levels and activity. Metabolites can also act as signaling molecules that influence gene expression
• What is the role of fructose 2,6-bisphosphate in glycolysis and gluconeogenesis?

  Fructose 2,6-bisphosphate activates phosphofructokinase-1 (PFK-1) to promote glycolysis and inhibits fructose 1,6-bisphosphatase to suppress gluconeogenesis, thus acting as a key regulator of glucose metabolism
• What happens during the phosphorylation of glycogen synthase?

  Phosphorylation of glycogen synthase by various kinases leads to its inactivation, thereby inhibiting glycogenesis
• What is the mass action ratio (MAR) and how is it used to identify rate-limiting steps?

  MAR is the ratio of product to substrate concentrations within a cell. To identify rate-limiting steps, compare MAR with the equilibrium constant (Keq). If MAR is significantly lower than Keq, the reaction is likely rate-limiting
• What is the role of hexokinase in glycolysis and how is it regulated?

  Hexokinase catalyzes the phosphorylation of glucose to glucose-6-phosphate in glycolysis. It is regulated by product inhibition, where glucose-6-phosphate inhibits hexokinase to prevent excessive glucose phosphorylation
• How does phosphofructokinase-1 (PFK-1) regulate glycolysis?

  PFK-1 is a key regulatory enzyme in glycolysis, activated by AMP and fructose 2,6-bisphosphate, and inhibited by ATP and citrate, thus controlling the flux of glucose through glycolysis based on the cell’s energy needs
• What is the role of citrate in the regulation of metabolic pathways?

  Citrate inhibits PFK-1 in glycolysis and signals sufficient energy supply, thus redirecting glucose towards storage or other pathways
• Explain the concept of a crossover point in metabolic regulation

  A crossover point occurs when perturbations in the system, such as hormonal stimulation, change the activity of a rate-limiting enzyme, increasing product concentration and decreasing substrate concentration, indicating the step’s regulatory importance
• Describe the regulatory effect of AMP on glycolysis and gluconeogenesis

  AMP activates PFK-1 in glycolysis to promote ATP production and inhibits fructose 1,6-bisphosphatase in gluconeogenesis, thus favoring energy production when cellular energy is low
• How does insulin affect glycogen metabolism?

  Insulin promotes glycogen synthesis by activating glycogen synthase through dephosphorylation and inhibits glycogen breakdown by reducing the activity of glycogen phosphorylase
• Explain how adrenaline affects glycogen metabolism

  Adrenaline increases glycogenolysis by activating glycogen phosphorylase through phosphorylation and inhibits glycogen synthesis by inactivating glycogen synthase, providing quick energy during stress
• What is the role of pyruvate kinase in glycolysis and how is it regulated?

  Pyruvate kinase catalyzes the final step in glycolysis, converting phosphoenolpyruvate to pyruvate. It is activated by fructose 1,6-bisphosphate and inhibited by ATP and alanine, balancing energy production
• How does phosphorylation control the activity of enzymes in metabolic pathways?

  Phosphorylation alters enzyme conformation, either activating or inactivating the enzyme, providing a rapid and reversible means of regulating metabolic pathways in response to hormonal signals
• Describe the slow extrinsic control of enzyme activity via hormones

  Slow extrinsic control involves changes in gene expression induced by hormones, leading to increased or decreased synthesis of specific enzymes, thereby modulating metabolic pathways over a longer period
• What is the importance of fructose 2,6-bisphosphate in regulating glucose metabolism?

  Fructose 2,6-bisphosphate is a potent activator of PFK-1, enhancing glycolysis, and an inhibitor of fructose 1,6-bisphosphatase, suppressing gluconeogenesis, thereby ensuring a balanced glucose metabolism
• How do glucagon and adrenaline affect gluconeogenesis and glycolysis?

  Both glucagon and adrenaline stimulate gluconeogenesis and inhibit glycolysis by promoting the phosphorylation and activation of enzymes involved in gluconeogenesis and deactivating glycolytic enzymes
• What is product inhibition and give an example?

  Product inhibition is when the product of an enzymatic reaction inhibits the enzyme that produced it. For example, glucose-6-phosphate inhibits hexokinase