Advanced Clinical Chemistry - Enzyme Principles and Kinetics

General Principles of Enzymes

  • Enzymes are biological catalysts found in all cells, catalyzing specific reactions.

  • They supply energy and/or chemical changes necessary for vital activities, enhancing reaction rates by factors of 10^6 to 10^{12}.

  • Enzymes are proteins produced under the control of specific genes, and they have four structural levels: primary, secondary, tertiary, and quaternary.

  • Enzymes are not altered or consumed in chemical reactions. Only small amounts are needed as they can be reused.

  • They accelerate the speed at which a chemical reaction reaches equilibrium without altering the equilibrium constant (amount of product from substrate).

Enzymatic Structures

  • Apoenzyme: Heat-labile protein part requiring a coenzyme.

  • Holoenzyme: Active enzyme consisting of the apoenzyme and cofactor/coenzyme.

  • Cofactors: Non-protein molecules required for enzyme function (can be organic or inorganic).

  • Activators: Inorganic cofactors such as metal ions (Fe^{2+}, Cl^{-}, Mg^{2+}).

  • Coenzymes: Organic cofactors related to vitamins (e.g., NAD), where bound coenzymes are termed prosthetic groups.

  • Proenzyme/Zymogen: Inactive enzyme forms secreted from the organ of production (like digestive enzymes).

  • Isoenzyme: Different forms of enzymes based on properties such as electrophoretic mobility and solubility.

Enzyme Classification

  1. Oxidoreductases: Catalyze oxidation-reduction reactions (e.g., LDH, G6PD).

  2. Transferases: Catalyze transfer of functional groups (e.g., AST, ALT, CK, GGT).

  3. Hydrolases: Catalyze hydrolysis of bonds (e.g., ALP, ACP, AMY, LPS).

  4. Lyases: Catalyze removal of groups without hydrolysis, creating double bonds (e.g., ALD).

  5. Isomerases: Catalyze interconversion of isomers.

  6. Ligases: Join two substrates, breaking the pyrophosphate bond in ATP or similar compounds.

Enzyme Kinetics and Reaction Dynamics

  • Energy of Activation (EA): Energy needed to form the activated complex from one mole of substrate.

  • The relationship among enzyme (E), substrate (S), and product (P):

E + S \rightleftharpoons ES \rightleftharpoons E + P

Factors Influencing Enzymatic Reactions

  • Substrate Concentration: Reaction rate increases to a certain point with increased substrate.

  • Enzyme Concentration: Higher enzyme concentration increases rate.

  • Temperature: Most enzymes have optimal velocities at 37°C; denaturation occurs above 40-50°C.

  • pH: Enzymes have optimal pH ranges, usually between 7-8, with exceptions (ACP, ALP).

  • Cofactors: Activators and coenzymes can increase reaction rates.

  • Inhibitors: Decrease reaction rates.

Enzyme Measurement Units

  • Enzymes are measured by activity rather than mass. Standard methods detect changes in product or substrate concentration.

  • International Unit (IU): Defined as the amount of enzyme that produces 1 μmol of product per minute under standard conditions.

Detailed Kinetic Parameters

  • Vmax: Maximum reaction rate when all enzyme molecules are bound to substrate.

  • Michaelis-Menten Constant (Km): Substrate concentration when initial velocity is ½ Vmax; it is specific for enzyme-substrate pairs.

  • Km: Indicates the affinity of the enzyme for its substrate; lower Km values suggest higher affinity, while higher Km values indicate lower affinity.

  • Lineweaver- Burk plot: A double-reciprocal plot used to determine Km and Vmax by plotting 1/v against 1/[S]; the slope of the line gives Km/Vmax, and the y-intercept represents 1/Vmax.

  • Fixed time: A method where the reaction time is held constant, allowing for the measurement of product formation at specific intervals to assess enzyme activity.

  • Continuous monitoring or kinetic assay: A method where the change in substrate concentration or product formation is measured continuously over time, providing real-time data on enzyme activity and allowing for the calculation of kinetic parameters such as Km and Vmax.

  • Lag Phase: A period of time at the beginning of the enzymatic reaction when the rate of product formation is initially low as the enzyme and substrate molecules undergo initial binding and adjustment before reaching a steady state where the reaction velocity accelerates.

  • Linear Phase: This phase occurs after the lag phase and is characterized by a constant rate of product formation, indicating that the enzyme is operating at its maximum efficiency under the given conditions.

  • Substrate depletion phase: This phase follows the linear phase and is marked by a decrease in the rate of product formation, as the availability of substrate becomes limited, leading to a reduction in enzyme activity and eventual saturation.

Types of Kinetics

  • First-order kinetics: Reaction velocity is proportional to substrate concentration.

  • Zero-order kinetics: Reaction rate is independent of substrate concentration.

Enzyme Inhibition Types

  • Competitive Inhibition: Inhibitor resembles substrate, competing for the active site (reversible). Km increased.

  • Noncompetitive Inhibition: Inhibitor binds to an allosteric site, altering the enzyme's function (can be reversible or irreversible). VMAX decreased.

  • Uncompetitive Inhibition: Inhibitor binds to the ES complex, preventing product formation. Both VMAX and Km decreased.

Diagnostic Enzymology

  • Enzyme assays typically measure product formation or substrate depletion, not enzyme concentration.

  • Enzymes can serve as reagents for measuring other non-enzymatic serum constituents.

  • Abnormal enzyme levels in serum may indicate various diseases; e.g., increased ALT in viral hepatitis, increased ALP during osteoblastic activity, increased LDH in hemolytic conditions.

  • Isoenzyme concentrations can help identify specific conditions (e.g., CK-BB for brain injury).

  • Hemolysis can affect enzyme analysis; serum must be separated from RBCs promptly to avoid contamination.