Enzyme Kinetics and Reaction Mechanisms

Enzymes

• Enzymes are biological catalysts, meaning they speed up chemical reactions without being consumed or changed by the reaction.
• Almost all enzymes are proteins, but not all proteins are enzymes.
• Some RNA molecules (ribozymes) can also act as enzymes.
• Life as we know it would not exist without enzymes.
• Enzymes consist of 100-300 amino acids linked into unbranched polypeptide chains.
• Substrate + Enzyme → Product + Enzyme

Enzyme Nomenclature

• Old System: Enzymes were initially named based on their function, often related to degradation reactions. The suffix "-ase" was added to the name of the substrate.
  • Example: Arginase hydrolyzes arginine to urea and ornithine.
  • Example: Urease hydrolyzes urea to ammonia and carbon dioxide.
• New System (EC numbers): The International Union of Biochemistry and Molecular Biology (IUBMB) introduced a standardized system using EC numbers (Enzyme Commission numbers).
  • These numbers are 4 digits long.
  • The first digit indicates the class of the enzyme.
  • The next two digits indicate the subclass.
  • The fourth digit indicates the specific enzyme within the subclass.

Enzyme Classification

• Oxidoreductases: Catalyze oxidation-reduction reactions (e.g., lactate dehydrogenase).
• Transferases: Catalyze the transfer of a functional group (e.g., methyl transferase).
• Hydrolases: Catalyze the hydrolysis of a bond (e.g., urease).
• Lyases: Catalyze the cleavage of a bond (e.g., decarboxylase).
• Isomerases: Catalyze the isomerization of a molecule (e.g., methylmalonyl CoA mutase).
• Ligases: Catalyze the joining of two molecules (e.g., pyruvate carboxylase).

Structure of Enzymes

• Apoenzyme: The protein component of a holoenzyme.
• Cofactors: Metal ions (e.g., Zn2+, Fe2+) that bind in a transient, dissociable manner.
• Coenzymes: Organic molecules that bind to the apoenzyme and participate in catalysis. They often derive from vitamins.
  • Cosubstrates are coenzymes that only associate temporarily with the enzyme.
  • Prosthetic groups are coenzymes that associate with the enzyme permanently.
• Holoenzyme: The complete, active enzyme consisting of the apoenzyme and cofactor/coenzyme (e.g., E + cofactor).
• Active site: The specific region of the enzyme where the substrate binds. The active site has a 3-D structure resulting from specific amino acid interactions.
• Isoenzymes: Different forms of an enzyme that have slightly different structures but similar functions. These variations may result from encoded by different genes and have different kinetic or regulatory properties (e.g., LDH, CK).
• Zymogens (proenzymes): Inactive precursor forms converted to active enzymes through proteolytic activation, which is a mechanism for activating proteins outside cells.
  • Example: Trypsinogen → Trypsin
  • Example: Pepsinogen → Pepsin

Enzyme Kinetics

• Transition state: A high-energy intermediate formed during the conversion of reactants to products. Enzymes lower the activation energy to speed up reactions.
• Rate of reaction: The higher the enzyme concentration, the greater the reaction velocity.

Michaelis-Menten Kinetics

• Michaelis-Menten equation: Describes how the initial reaction velocity (v°) varies with substrate concentration ([S]).
  • v° = (Vmax[S]) / (Km + [S])
  • Vmax is the maximal velocity.
  • Km is the Michaelis constant.
• Order of reaction: When [S] is much less than Km, reaction is first order; When [S] is much greater than Km, reaction is zero order

Lineweaver-Burk Plot

• This linear plot of 1/v0 vs. 1/[S] allows for easier determination of Vmax and Km values.

Enzyme Inhibition

• Irreversible inhibitors: Bind covalently to the enzyme, permanently inactivating it (e.g., DIFP, nerve gases).
• Reversible inhibitors: Bind noncovalently to the enzyme and can be removed, leading to different effects depending on the type of inhibitor.
  • Competitive inhibitors: Structurally similar to the substrate; compete with the substrate for binding to the active site. Increased substrate concentration can overcome competitive inhibition.
  • Noncompetitive inhibitors: Bind to a site other than the active site, altering the enzyme's conformation. These inhibitors cannot be overcome with increased substrate.
  • Uncompetitive inhibitors: Bind only to the enzyme-substrate complex, at a location other than the active site.

Regulation of Enzyme Activity

• Allosteric regulation: Involves allosteric effectors which bind non-covalently to allosteric sites, altering the enzyme's shape and activity. The binding of one substrate can affect the binding of another substrate.
• Covalent modification (e.g., phosphorylation/dephosphorylation). Addition/removal of chemical groups.
• Enzyme synthesis and breakdown: Increased or decreased synthesis, or degradation.

Clinical Applications

• Clinical use: measuring enzyme activity in the blood to diagnose diseases or monitor therapy.
  • Elevation of specific enzymes in the blood can indicate specific disorders. Enzyme activity (e.g., AST, ALT) can help in monitoring the efficacy of therapy for liver diseases or other metabolic disorders.