enzymes

Basic Principles of Enzymes

• Definition: Enzymes are biological catalysts that facilitate chemical reactions in organic matter, primarily within cells.

  • They can catalyze a single or limited number of chemical reactions.

• Functionality:

  • Enzymes are large molecules confined to cells, participating in intracellular processes.

  • Increased membrane permeability allows enzymes to access blood plasma, especially after cellular injury.

• Measurement:

  • Enzymes are measured by their activity rather than their absolute concentration.

  • An example is amylase, which is measured by the products of its enzymatic reactions, not by the enzyme itself.

• Clinical Significance:

  • Enzymes appear in serum following cellular injury; their presence can indicate organ damage.

  • They react with specific substrates to produce defined products.

• Active and Allosteric Sites:

  • Active Site: Where substrate binding occurs, enabling the reaction.

  • Allosteric Site: A site where regulatory molecules bind, affecting enzyme activity.

Factors Affecting Enzymatic Reactions

• Enzyme Concentration:

  • Higher enzyme concentrations lead to faster reactions, demonstrating a proportional relationship.

• Substrate Concentration:

  • The reaction rate increases with substrate concentration until it reaches a saturation point.

  • Beyond saturation, adding more substrate does not increase the reaction rate.

• Cofactors:

  • Coenzymes: Non-protein substances necessary for enzyme activity (e.g., NAD, NADP).

  • Apoenzyme: The inactive protein component of the enzyme.

  • Holoenzyme: The active form of the enzyme (apoenzyme + cofactor/coenzyme).

• Inhibitors:

  • Competitive Inhibitors: Compete with the substrate for the active site; their effects are reversible.

  • Noncompetitive Inhibitors: Bind to the allosteric site, changing the active site and decreasing activity irreversibly.

  • Uncompetitive Inhibitors: Bind to the enzyme-substrate complex, inhibiting the reaction.

• Isoenzymes:

  • Variants of enzymes that catalyze the same reaction but differ slightly in structure (e.g., lactate dehydrogenase with multiple isoenzymes).

• Temperature:

  • Enzymes have optimum activity at around 37°C (body temperature).

  • Temperature changes can denature enzymes or alter their activity.

• pH Levels:

  • The optimum pH for enzymatic activity is generally between 7 and 8.

  • Extreme pH values can lead to enzyme denaturation.

• Storage Conditions:

  • Enzymes can be reversibly inactive when refrigerated but may denature upon repeated freeze-thaw cycles.

• Hemolysis and Specimen Quality:

  • Hemolysis releases intracellular enzymes into the serum, artificially raising enzyme levels in tests.

  • Lipemic samples (presence of increased triglycerides) can decrease enzyme concentration on analysis.

Enzyme Nomenclature and Classification

• Standardization:

  • Enzymes are named and classified by the Enzyme Commission (EC), a system established in 1961 and revised thereafter.

  • Each enzyme has a unique EC number based on its biochemical function and reaction type.

• Classes of Enzymes:

  1. Oxidoreductases: Catalyze redox reactions.

  2. Transferases: Transfer chemical groups between molecules.

  3. Hydrolases: Catalyze hydrolysis reactions (breaking bonds with water).

  4. Lyases: Break bonds without hydrolysis.

  5. Isomerases: Catalyze rearrangements of molecules.

  6. Ligases: Join two substrate molecules.

Enzyme Kinetics and Detection Methods

• Michaelis-Menten Kinetics:

  • Describes how reaction rates change with substrate concentration.

  • Initially, increased substrate concentration leads to increased reaction rates until saturation.

  • Reaction velocity plateaus once maximum enzyme activity is reached.

• Kinetics Order:

  • Zero Order: Reaction rate is independent of substrate concentration, often determined by enzyme concentration.

  • First Order: Reaction rate depends on substrate concentration; decreases as substrate is consumed.

• Assay Methods:

  • Fixed time and kinetic/continuous monitoring assays to measure enzyme activity.

  • Units for enzymatic activity include International Units (IU) and Catalytic Units (Katal).

Clinical Applications and Causes of Elevated Enzyme Levels

• Elevated Plasma Enzymes:

  • Impaired renal function can reduce enzyme clearance from the bloodstream.

  • Increased membrane permeability or cell turnover can result in excess enzyme leakage.

  • Tissue damage, necrosis, or other pathological conditions can elevate serum enzyme levels.