Clinical Chemistry I - Week 12 Quiz

Clinical Chemistry I - Week 12: Enzyme Properties and Reaction Influencing Factors

• Course: Clinical Chemistry I
• Course Code: 0202304
• Instructor: Mohammad Qabajah
• Textbook: Tietz Fundamentals of Clinical Chemistry and Molecular Diagnostics (8th ed.) by Rifai, Horvath, & Wittwer (2019)

Objectives

• Define enzymes and their structural components.
• Discuss enzyme characteristics, naming, and classification.
• Explain enzyme activity and influencing factors.
• Compare substrate-enzyme complex formation theories.
• Differentiate first-order and zero-order kinetics.
• List clinically significant enzymes by name and abbreviation.
• Explain enzyme testing methods (fixed time and continuous monitoring).
• Discuss simplified enzyme activity measurement procedures.
• Identify abnormal enzyme results (low, normal, high) and possible diagnoses based on results.

Definition of the Enzyme

• Large protein substances in cells (catalysts, not consumed).
• Highly specific for their substrate.
• Optimal conditions for activity (temperature, pH, etc.).
• 2,000-3,000 different enzymes in each cell.
• Involved in multi-step biochemical pathways.
• Regulated through feedback control.
• Isoenzymes (isozymes) catalyze the same reactions but differ structurally.
• Production directly controlled by genes; genetic diseases can affect enzyme systems.

Enzyme Nomenclature

• Standardized by the Enzyme Commission (EC) of the International Union of Biochemistry (IUB).
• Suffix "ase" added to the name (e.g., Lactase).
• Suffix "in" added when the compound is digested (e.g., Pepsin).
• Each enzyme has a recommended name, abbreviation, and EC code number.

Enzyme Composition and Structure

• Enzymes are distinguished from other proteins by their catalytic activity.
• Any structural change can affect catalytic activity.
• Holoenzyme: Complete enzyme with all parts.
  • Apoenzyme: Protein portion of the enzyme.
  • Cofactor: Non-protein substance (activator or coenzyme).
• Catalytic Site (Active Site): Region that binds the substrate.
• Substrate: Substance acted upon by the enzyme in a chemical reaction.
• Allosteric Site: Enzyme cavity not the active site, often for regulation.

Enzyme Cofactors

• Non-protein substances help enzymes reach maximal activity.
• Two types of cofactors:
  • Inorganic cofactors (activators): Examples: chloride ions, magnesium ions.
  • Organic cofactors (coenzymes): Derived from vitamins; examples: NAD, NADH, NADP, NADPH.
  • Prosthetic group: Organic cofactors tightly bound to the enzyme.

Enzyme Reaction: Example

• L-lactate + NAD → Pyruvate + NADH + H+ (Enzyme: LDH)
  • Substrate: L-lactate
  • Cofactor (Coenzyme): NAD
  • Product 1: Pyruvate
  • Product 2: NADH + H+

Enzyme Catalytic Properties

• Enzymes are catalysts.
• How enzymes work
  • Lower the activation energy for reactions.
  • Increase reaction rate without being consumed.
  • Usually catalyze between 1-10,000 substrate molecules per second.

Factors Affecting Enzyme Action

• Temperature: Optimum activity near physiological temperature (e.g., 37°C in humans). High temperatures denature enzymes.
• pH: Optimum activity within a narrow pH range (physiological pH). Extreme pH values denature enzymes.
• Enzyme Concentration: Reaction rate dependent on enzyme concentration (zero-order kinetics). Enzyme concentration limited by substrate concentration.
• Substrate Concentration: Reaction rate increases with substrate concentration (first-order kinetics).
• Inhibitors: Cause a loss of enzyme activity.
  • Competitive: bind to active site.
  • Non-competitive: bind to different site.
  • Uncompetitive: bind to enzyme-substrate complex. Inhibitor is useful for medication design.

Testing of Enzymes

• Enzymes are not a health problem but rather a marker of cell death.
• Enzymes are present in small amounts of fluids and are used to diagnose diseases.
• Easier to measure catalytic activity relative to concentration.

###Testing of Enzymes: Enzyme Activity Measurement

• Procedure based on the reaction the enzyme catalyzes.
  • Add substrate to the enzyme sample in a controlled environment (optimum temp, pH).
  • Incubate.
  • Measure products to quantify enzyme activity (using Spectrophotometer).
• Fixed-Time Method: Measures reaction amount after a fixed time.
• Continuous Monitoring Method (Kinetic): Measurements taken during the reaction.

Enzyme Units and Calculation

• Units defined as catalyst reaction speeds (e.g., international units – IU).
• Enzymes usually expressed in units per liter (IU/L or U/L).

Enzymes as Reagents

• Enzymes used to measure other constituents.
• Example: glucose measurement using hexokinase enzyme.

Clinically Significant Enzymes

• List of clinically significant enzymes important in diagnoses.
  • Liver enzymes (AST, ALT, ALP).
  • Cardiac enzymes (CK-MB, LDH).
  • Others (Amylase, Lipase, Acid phosphatase, etc.).