Principles of Enzymatic Catalysis and Biocatalysis

Principles of Enzymatic Catalysis

• Enzymes act as catalysts in biochemical processes.
• They increase reaction rates without being consumed in the process.
• Enzymes accelerate both the forward and reverse reactions, driving systems towards equilibrium faster.

Advantages of Biocatalysis

• Greater reaction specificity: Enzymes minimize the formation of unwanted side products.
• Milder reaction conditions: Enzymes operate effectively in the physiological environment of cells, at pH ~ 7 and 37°C.
• Higher reaction rates: Enzymes allow for efficient biochemical processes within the timescale of biological systems.
• Regulation and control: Enzymes play a crucial role in regulating and controlling metabolic pathways.

Enzymes in Disease

• Enzyme levels and activities can serve as biomarkers for diagnosing diseases.
• Changes in enzyme concentrations or activities are indicative of specific disease states.
• Many medications target and interact with specific enzymes.

Enzyme Structure and Function

• Most enzymes are globular proteins, with catalytic activity dependent on their native folded structure, including primary, secondary, tertiary, and quaternary structures.
• Some RNA molecules, like ribozymes and ribosomal RNA, have catalytic properties.
• Prosthetic groups, such as cofactors (inorganic ions) and coenzymes (organic molecules), can be critical for enzymatic activity.
  • Apoenzyme: the protein component of an enzyme without its prosthetic group
  • Holoenzyme: the active complex formed by the apoenzyme and its prosthetic group(s).

Enzyme Classification

• The International Classification of Enzymes (ICE) categorizes enzymes into six classes based on their catalytic activity:
  • Oxidoreductases: Catalyze electron transfer reactions.
  • Transferases: Transfer functional groups between molecules.
  • Hydrolases: Break bonds through the addition of water.
  • Lyases: Cleave bonds by mechanisms other than hydrolysis or oxidation.
  • Isomerases: Rearrange atoms within a molecule.
  • Ligases: Join two molecules by forming new bonds, often requiring ATP or cofactors.

Enzyme Substrate Selectivity

• Enzymes exhibit high specificity for their substrates, binding to specific molecules while excluding others.
• This selectivity arises from the complementary fit between the enzyme's active site and the substrate.
• Example: phenylalanine hydroxylase specifically acts on phenylalanine.
• Enzyme-Substrate Complex: The binding of a substrate to an enzyme's active site forms a crucial intermediate complex, facilitating the catalytic process.

How Enzymes Lower Activation Energy

• Transition State Stabilization: Enzymes bind to the transition state of a reaction more strongly than they bind to the substrate.
  • This stabilization lowers the activation energy (ΔG‡), accelerating the reaction.
• Proximity Model: The close proximity of reactive groups within the active site enhances their interaction, leading to faster reaction rates.

Catalytic Mechanisms of Enzymes

• Common mechanisms employed by enzymes:
  • Acid-Base Catalysis: Amino acid residues donate or accept protons to facilitate proton transfer.
  • Covalent Catalysis: Transient covalent bonds form between the enzyme and the substrate, changing the reaction pathway.
  • Metal Ion Catalysis: Metal ions are incorporated into the active site for various roles, including:
    • Stabilizing negative charges
    • Facilitating substrate binding
    • Participating in redox reactions

Chymotrypsin: A Case Study

• Chymotrypsin is a protease involved in protein digestion.
• It cleaves peptide bonds adjacent to aromatic amino acids.
• Chymotrypsin effectively uses multiple catalytic mechanisms, including acid-base, covalent, and metal ion catalysis.

Enzyme Kinetics

• Kinetics: Study of the rates of chemical reactions.
• Factors influencing enzymatic reaction rates:
  • Enzyme concentration
  • Substrate concentration
  • Effectors (activators or inhibitors)
  • Temperature
• Applications of enzyme kinetics:
  • Quantitative description of enzymatic catalysis
  • Elucidation of substrate binding order
  • Understanding catalytic mechanisms
  • Discovery of effective inhibitors
  • Investigation of enzyme activity regulation

Enzyme Inhibition:

• Irreversible Inhibitors (inactivators): permanently inactivate the enzyme, forming a strong complex.
• Reversible Inhibitors: bind to and dissociate from the enzyme, slowing down its activity.
• Reversible inhibitor types:
  • Competitive: Inhibitor and substrate compete for binding to the active site.
  • Uncompetitive: Inhibitor binds only to the enzyme-substrate complex (ES).
  • Mixed: Inhibitor binds to the enzyme with or without substrate, affecting both substrate binding and catalysis.

Effects of Inhibition on Enzyme Kinetics:

• Competitive Inhibition:
  • No change in Vmax (maximum velocity)
  • Apparent increase in Km (Michaelis-Menten constant, representing the substrate concentration at half Vmax)
  • Lineweaver-Burk plot: lines intersect at the y-axis.
• Uncompetitive Inhibition:
  • Decrease in Vmax
  • Apparent decrease in Km
  • No change in Km/Vmax
  • Lineweaver-Burk plot: lines are parallel.
• Mixed Inhibition:
  • Decrease in Vmax
  • Apparent change in Km
  • Lineweaver-Burk plot: lines intersect left to the y-axis.