Enzyme Regulation Notes

Enzyme Regulation

Enzymes and Regulation

  • Enzyme regulation, Hemoglobin's oxygen transport, covalent modification, proteolytic cleavage, and allosteric inhibition of aspartate transcarbamoylase (ATCase) are key topics.

Metabolic Pathways

  • Metabolic pathways involve multiple enzyme steps within a single pathway.
  • The glycolytic pathway oxidizes sugars.
  • Regulation of a pathway is achieved by regulating one key enzyme.
  • Inhibition of the first enzyme in a pathway can shut down the entire pathway.

Feedback Inhibition

  • Feedback inhibition: the end-product of a pathway inhibits the first unique step in its synthesis.
  • This regulates the product's concentration within the cell.
  • Example: Molecule Z inhibits the conversion of molecule B to molecule X.

Physiological Regulation of Enzyme Activity

  • Mechanisms:
    • Allosteric control: using non-substrate small molecules as modulators.
    • Reversible covalent modification: e.g., phosphorylation/dephosphorylation for hormonal regulation.
    • Proteolytic activation: activating digestive enzymes in the gut and blood-clotting cascade after injury.

Allosteric Regulation

  • Allosteric proteins have regulatory and active sites.
  • Signal molecules bind to sites distant from the active site, causing conformational changes that affect the active site.
  • Allosteric proteins show cooperativity: activity at one site affects others.
  • Examples: ATCase and hemoglobin.

ATCase: End-Product Inhibition

  • ATCase catalyzes the first step in pyrimidine synthesis.
  • It is inhibited by CTP, an end-product of the pathway, demonstrating end-product or feedback inhibition.

Allosteric Inhibition of ATCase

  • CTP binds to a regulatory site separate from the catalytic site.
  • ATCase has catalytic trimers linked by regulatory chain dimers.

Kinetics of Allosteric Enzymes

  • Allosteric enzymes do not follow Michaelis-Menten kinetics; they show sigmoidal kinetics.
  • Cooperativity: Substrate binding increases binding properties in other subunits.
  • Isolated catalytic subunits follow normal kinetics; regulatory subunits cause the sigmoidal curve.

Allosteric Effects and Shape Changes

  • ATCase exists in two conformations:
    • Tense (T) state: compact and less active.
    • Relaxed (R) state: expanded and more active.
  • Substrate binding favors the R state, while CTP binding favors the T state.
  • Cooperativity: Ligand binding to one subunit affects the shape of others.

Hemoglobin and Cooperativity

  • Hemoglobin: a tetrameric protein transporting oxygen.
  • Cooperativity allows hemoglobin to deliver more oxygen to tissues.
  • Partial pressure of oxygen (pO_2) in the lungs is ~100 torr, and in tissues, it is ~20 torr.
  • Oxygen delivery depends on the difference in hemoglobin saturation at these pO_2 levels.

2,3-BPG Modulation of Hemoglobin

  • 2,3-BPG is present in red blood cells.
  • It stabilizes the T state of deoxyhemoglobin, reducing oxygen affinity.
  • This facilitates oxygen release in target tissues.

Fetal Hemoglobin

  • Fetal hemoglobin: α2γ2 instead of adult α2β2.
  • Lower affinity for 2,3-BPG due to the γ_2 subunits.
  • Ensures efficient oxygen transfer from maternal to fetal red blood cells.

Regulation via Covalent Modification

  • Reversible covalent modification regulates enzyme activity.
  • Phosphorylation/dephosphorylation is the most common.
  • Other modifications include acetylation of histones and lipid additions for membrane anchoring.

Protein Kinases

  • Protein kinases phosphorylate proteins by transferring a phosphate group from ATP to serine, threonine, or tyrosine residues.
  • There are >500 human protein kinases.

Protein Phosphatases

  • Protein phosphatases remove phosphate groups from phosphorylated proteins, releasing inorganic phosphate (P_i).
  • Phosphorylation status depends on the relative activities of kinases and phosphatases.

Phosphorylation and Protein Structure

  • Phosphorylation alters substrate binding and catalytic activity.
  • The phosphoryl group ( -OPO_3^{2-} ) introduces negative charges and promotes hydrogen bonds.
  • The energy from the phosphate bond can shift equilibrium between protein structures.

Phosphorylation and Enzyme Activity

  • Some enzymes are inactivated by phosphorylation (e.g., glycogen synthase).
  • Others are activated (e.g., glycogen phosphorylase).

Kinase-Phosphatase Cycle

  • Phosphorylation and dephosphorylation are reversible, occurring rapidly or over hours.

Regulation of Protein Kinase Activity

  • Protein kinase A (PKA) is a key enzyme in hormone-regulated enzyme activation.
  • Activated by epinephrine and glucagon via cyclic AMP (cAMP).
  • cAMP, a second messenger formed from ATP cyclization, activates PKA, which then phosphorylates intracellular enzymes.

Activation of Protein Kinase A

  • PKA has regulatory (R) and catalytic (C) subunits.
  • The pseudosubstrate portion of R blocks catalytic sites.
  • cAMP binding to R subunits releases the catalytic subunits, allowing them to phosphorylate substrate proteins.

Proteolytic Activation of Enzymes

  • Some enzymes are synthesized as inactive zymogens or proenzymes, activated by cleavage of specific peptide bonds.
  • Activation is irreversible.
  • Used for digestive enzymes and peptide hormones like insulin.

Chymotrypsinogen to Chymotrypsin

  • Chymotrypsin is initially synthesized as chymotrypsinogen in the pancreas.
  • Trypsin cleaves a peptide bond in the small intestine.
  • The resulting π-enzyme cleaves itself to form the α form. Both are active.
  • The separated chains stay linked by disulfide bonds.

Inactivity of Chymotrypsinogen

  • Chymotrypsinogen is an inactive zymogen.
  • Cleavage forms π-chymotrypsin, causing conformational changes.
  • The new amino terminal isoleucine 16 forms an ionic bond with aspartate, stabilizing the active site.
  • Without these changes, the enzyme is inactive.

Zymogen Activation of Digestion

  • Enteropeptidase, produced in the duodenum, activates trypsinogen to trypsin.
  • Trypsin then activates other pancreatic zymogens.
  • Secretion as zymogens prevents autodigestion of the pancreas.