Enzymes

Introduction to Enzymes

  • Enzymes are:

    • Specific biologic proteins.

    • Catalysts for biochemical reactions.

    • Characteristics:

    • Do not alter the equilibrium point of the reaction.

    • Are not consumed in the reaction.

    • Do not change in composition.

  • Importance of Reactions:

    • Necessary for physiological function.

    • Present in all body tissues.

    • Serum enzyme levels increase after cellular injury.

    • May be detectable in serum after cell degradation.

Protein Structure

  • Types of Protein Structures:

    • Primary Structure:

    • Sequence of a chain of amino acids.

    • Secondary Structure:

    • Formation when amino acids are linked by hydrogen bonds; examples include alpha helix and beta pleated sheet.

    • Tertiary Structure:

    • Formed through attractions between alpha helices and pleated sheets, creating a 3D shape.

    • Quaternary Structure:

    • Consists of more than one amino acid chain.

Enzyme Structure

  • 3D Structure properties:

    • Twisting and folding of the protein chain creates specific active sites.

    • Chemistry and shape of the active site determine substrate binding:

    • The active site and substrate must match (specificity).

Terms Relating to Enzyme Specificity

  • Specificity Types:

    • Absolute Specificity:

    • Enzyme catalyzes only one type of reaction with one known substrate.

    • Group Specificity:

    • Enzyme acts on various substrates sharing common chemical groups.

    • Bond Specificity:

    • Refers to the specific interactions with specific types of bonds.

    • Stereoisometric Specificity:

    • The ability to bind with only one optical isomer of a compound.

Terms Related to Enzymes

  • Active Site:

    • Region where the substrate interacts, typically devoid of water.

  • Co-factor:

    • A non-protein molecule that binds to an enzyme, necessary for activity.

    • Completes shape of active site or assists in substrate binding or catalysis.

    • Relationship: Inactive enzyme + cofactor = Active enzyme.

Co-factors

  • Types of Co-factors:

    • Activators:

    • Usually inorganic ions (e.g., Mg²⁺, Na⁺).

    • Some are necessary for enzyme activity; some enhance reactions.

    • Note on serum: Anticoagulants can bind necessary activating ions (Ca²⁺, Mg²⁺).

    • Coenzymes:

    • Large organic molecules (e.g., vitamins).

    • Function as co-substrates, altering during the reaction.

    • Example with NAD:

      • NAD + H₂ (reduced substrate) → NADH + H⁺ (oxidized substrate).

      • Measures at 340 nm.

Enzyme Terms

  • Holoenzyme:

    • Complex formed from a prosthetic group and apoenzyme.

  • Proenzyme/Zymogen:

    • A precursor of the active enzyme in its inactive form.

  • Isoenzymes:

    • Various forms of an enzyme that differ in structure yet perform the same catalytic function, recognizable by:

    • Electrophoretic mobility.

    • Solubility.

    • Resistance to inactivation.

Enzyme Classification/Nomenclature

  • Traditional Naming:

    • Based on substrate and the suffix “-ase” (e.g., Lipase, Sucrase).

  • International Classification:

    • Systematic naming involves:

    • Substrate, reaction, possible coenzyme names.

    • Assigned EC numerical codes covering classes, subclasses, and serial numbers.

Classes of Enzymes

  • Oxidoreductases:

    • Catalyze oxidation-reduction reactions.

  • Transferases:

    • Move intact atoms or groups between molecules (e.g., NH₂).

  • Hydrolases:

    • Involve addition or removal of water molecules.

  • Lyases:

    • Split molecules without hydrolysis or oxidation.

  • Isomerases:

    • Catalyze inter-conversion of isomers.

  • Ligases:

    • Join two large substrates while breaking down a smaller molecule like ATP.

Enzyme Kinetics

  • Collision Theory:

    • Reactions occur when reactant molecules collide.

    • Reaction rates are proportional to the concentration of reactants.

    • Reactions need excess energy to proceed, usually provided by catalysts.

  • Activation Energy:

    • The energy required to initiate a reaction, which enzymes lower.

Enzyme-Substrate Binding

  • Process:

    • Ionic binding occurs between the enzyme and substrate.

    • Enzyme undergoes a conformational change, creating strain in substrate bonds, facilitating transformation.

    • General reaction representation:
      (E + S ↔ ES ↔ E + P)

    • E = enzyme, S = substrate, ES = enzyme-substrate complex, P = product.

Equilibrium in Enzymatic Reactions

  • Reaction can reach equilibrium:

    • Forward and reverse reactions are at the same rate.

  • Example with Carbonic Anhydrase:

    • (CO₂ + H₂O ↔ H₂CO₃ → H⁺ + HCO₃⁻).

Measurement of Enzymes

  • Reaction rate represents the amount of product formed over time.

    • Example: 10 µmol of product in 5 min equals a rate of 2 µmol/min.

  • Enzyme concentration directly correlates with reaction rate.

Factors Influencing Enzymatic Reactions: Substrate Concentration

  • First-Order Kinetics:

    • Rate dependent on substrate concentration.

    • If substrate concentration decreases, reaction rate also decreases.

  • Requirement for excess enzyme as reagent to ensure reactions proceed.

Factors: Enzyme Concentration

  • Zero-Order Kinetics:

    • When substrate concentration is high, all enzyme active sites are filled, leading to maximum reaction rate.

    • Reaction rate becomes dependent on enzyme concentration under saturated conditions.

Michaelis-Menten Constant

  • Km:

    • The substrate concentration at which the enzyme operates at half of its maximum velocity.

    • A specific constant for enzyme-substrate interactions under defined conditions.

Other Factors Influencing Enzymatic Reactions

  • pH Levels:

    • Enzymes have an optimal pH for activity (usually between 7.0 and 8.0).

    • Extreme pH changes can denature proteins.

  • Temperature:

    • Increasing temperature raises reaction rates until denaturation occurs (typically above 40-50°C for most enzymes).

  • Time:

    • Reactions progress until substrates are depleted; accurate timing is crucial for analyzing changes in enzyme activity.

Enzyme Inhibitors

  • Competitive Inhibitors:

    • Resemble substrate and bind to the active site, slowing the reaction rate.

    • Effect can be reduced by increasing substrate concentration.

  • Non-competitive Inhibitors:

    • Bind to enzymes at locations other than the active site, reducing reaction rate regardless of substrate concentration.

  • Uncompetitive Inhibitors:

    • Bind to ES complexes, preventing product formation; increased substrate can lead to more inhibition.

Enzymes as Analytical Reagents

  • Enzymes can be employed to measure analyte concentrations due to their specificity.

  • Reaction velocity correlates to substrate concentration determination.

    • Examples include glucose oxidase for glucose measurement.

Applications of Enzymes

  • Liquid Reagents:

    • Enzymes chemically bonded to adsorbents for stability.

  • Immunoassays:

    • Indicators in tests involving antigens or antibodies (e.g., horseradish peroxidase).

Measurement of Enzyme Activity

  • Enzymes present in small amounts but can elevate due to:

    • Increased cell destruction (e.g., MI, hepatitis).

    • Proliferation or demand for enzymes (e.g., tumors).

  • Measurement techniques include assessing product increase or substrate decrease.

Zero-Order Kinetics in Measurement

  • Essential to provide substrate, coenzyme, and reactants in excess to maintain zero-order kinetics.

    • Reaction rate becomes reliant solely on enzyme concentration.

Enzyme Measurement Techniques

  • Kinetic Assays:

    • Continuous absorbance monitoring; preferred for verifying linearity.

    • Used to mitigate early substrate depletions.

Calculating Enzyme Activity

  • Reported in Units (U/L or IU/L).

    • Defined as the enzyme amount catalyzing 1 μmole of substrate per minute under specified conditions.

    • Equation for enzyme activity includes absorbance measurements to determine activity and concentrations:

    • U/L = \frac{Δ \text{Absorbance} \times \text{total volume} \times 1 \times 10^6 \text{ μmol/mol}}{Δ \text{Time} \times \text{sample volume} \times ε \times b}.

Errors in Enzyme Measurement

  • Common errors involve:

    • Substrate depletion affecting reaction rates.

    • Improper sample storage leading to degradation or inaccurate results.

    • Presence of inhibitors or missing cofactors impacting enzyme activity.

Enzyme Methods and Quality Control

  • Immunoassays and electrophoresis utilized in enzyme concentration measurement.

  • Quality control issues can arise from variations in sample integrity and assay specificity.

Enzyme Measurements in Liver Disease

  • Specific enzyme measurements relevant to liver diseases include:

    • Alkaline Phosphatase (ALP): Increased in cholestasis and various liver abnormalities.

    • Aminotransferases (AST, ALT): Levels elevate in liver injury, most notably in acute conditions.

    • Lactate Dehydrogenase (LD): Used in assessing liver function across various conditions.

Measures of Alkaline Phosphatase Activity

  • ALP presence indicates various liver conditions and is influenced by structural elements of the enzyme.