Enzymes and Enzyme Regulation

Enzymes: Biological Catalysts

  • Enzymes are biological catalysts that speed up chemical reactions without being consumed or changed in the process.
  • Most enzymes are globular proteins, sharing structural features with other proteins (1°, 2°, 3°, and sometimes 4° structures).
  • A few enzymes are RNA molecules.

Enzyme Specificity and Reaction Rates

  • Enzymes act on specific substrates to produce products (E + F ⇌ A + B ⇌ C + D).
  • Enzymes offer:
    • Higher reaction rates.
    • Milder reaction conditions.
    • Greater reaction specificity.
    • Capacity for regulation due to conformational changes in protein structure and cooperativity.

How Enzymes Work

  • Enzymes function by affecting reaction rates, specifically speeding up reactions.
  • They achieve this by lowering the activation energy barrier (\Delta G^{\ddagger}).
  • Enzymes do NOT alter the free energy change of the reaction (\Delta G_{rxn}).

Activation Energy and Transition State

  • The speed of a biochemical reaction depends on the size of the activation energy barrier (\Delta G^{\ddagger} = G{TS} - GR).
  • The transition state (TS) represents the highest free energy state in the reaction.

Catalytic Mechanisms

  • Catalysts lower the activation energy barrier through:
    1. Desolvation: Removing substrates from aqueous solution.
    2. Proximity and orientation effects: Bringing reactants closer and in the correct orientation.
    3. Participation in the reaction mechanism.
    4. Stabilizing the transition state.
  • These mechanisms are mediated through the active site of the enzyme.

Active Site

  • The active site is a 3-D cleft/crevice within the protein's structure.

Substrate Binding

  • The active site's design contributes to:
    • Affinity: How well the enzyme binds the substrate.
    • Specificity: The enzyme's preference for a particular substrate.
  • Induced Fit Model: Substrate binding induces a conformational change in the enzyme, better resembling the transition state.

Desolvation

  • Sequestering substrates in a non-aqueous environment:
    • Prevents interference by water molecules.
    • Enhances H-bond formation.
    • Eliminates the energy barrier imposed by ordered solvent molecules.

Proximity and Orientation Effects

  • Enzymes increase reaction rates by bringing reactants into close proximity and proper orientation.

Participation in Reaction Mechanism

  • Enzymes position specific amino acid side chains in the active site to react with substrates through:
    • Acid-base catalysis.
    • Covalent/nucleophilic catalysis (forming a transient covalent bond between substrate and enzyme).
    • Metal ion catalysis (using cofactors).

Amino Acid Side Chains in Catalysis

  • Acid-Base Catalysis: Amino acid side chains (e.g., Glu, Asp, His, Cys, Tyr, Lys) act as acid or base catalysts depending on their protonation state.
  • Nucleophilic Catalysis: Deprotonated forms of amino acids (e.g., Ser, Tyr, Cys, Lys, His) act as nucleophiles. Asp and Glu can also participate.

Cofactors

  • Cofactors can be metal ions (e.g., Fe, Zn, Cu, K, Mg, Na) or coenzymes (organic molecules).
  • Coenzymes include cosubstrates (e.g., NAD+) and prosthetic groups (e.g., FAD).
  • Coenzymes must be regenerated after the reaction.

Transition State Stabilization

  • Enzymes bind the transition state better than the substrate, lowering its free energy through non-covalent interactions.
  • Transition state analogs are potent enzyme inhibitors, binding with higher affinity.

Regulating Enzyme Activity

  • Mechanisms for regulating enzyme activity in vivo:
    • Competitive inhibition.
    • Allostery.
    • Ionic signals.
    • Reversible covalent modification.
    • Regulation of gene expression (altering enzyme synthesis or degradation rates).
    • Changes in subcellular localization.

Competitive Inhibition

  • Inhibitors resemble the substrate and compete for binding to the active site.
  • Increase the apparent K_M (decrease affinity) between enzyme and substrate.

Allostery

  • Allosteric enzymes exhibit a sigmoidal relationship between reaction velocity and substrate concentration (homoallostery).
  • Undergo conformational changes upon effector binding (non-covalent).
  • Typically have quaternary structure and exhibit positive cooperativity.
  • Catalytic activity is modulated by non-covalent binding of specific molecules at a site other than the active site (heteroallostery).
  • Allosteric enzymes exist in two states: T (tense, low activity) and R (relaxed, high activity).
    • Allosteric inhibitors favor the T state.
    • Allosteric activators favor the R state.

Reversible Covalent Modification (Phosphorylation)

  • Involves the addition or removal of phosphate groups, often on Ser, Tyr, or Thr residues.
  • Kinases catalyze the transfer of a phosphate group from ATP to a protein.
  • Phosphatases catalyze the hydrolysis of a phosphate group from a molecule.
  • Phosphorylation results in:
    • Increased size and polarity/hydrophilicity.
    • Addition of two negative charges.
    • Capability of forming multiple new H-bonds.
  • Phosphorylation changes enzyme activity by modifying the protein’s 3-D shape.
  • Depending on the enzyme, phosphorylation may increase or decrease activity.