Enzyme activity and its measurement, physicochemical factors affecting enzyme activity, regulation of enzymes

Enzyme Activity and Its Measurement

a. Definition of Enzyme Activity

• Enzyme Activity: Rate of a reaction catalyzed by an enzyme.
• Specific Activity: Enzyme activity per milligram of protein (indicator of enzyme purity).
• Turnover Number (kcat): Number of substrate molecules converted per enzyme molecule per second.

b. Methods of Measurement

1. Spectrophotometric Assays: Measure changes in light absorbance (e.g., NADH at 340 nm).
2. Fluorometric Assays: Detect changes in fluorescence (high sensitivity).
3. Calorimetric Assays: Measure heat changes during enzymatic reactions.
4. Chemiluminescent Assays: Use light emission from reactions (e.g., luciferase-based assays).
5. Radioactive Assays: Track radioactive-labeled substrates or products (sensitive and specific).

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2. Physicochemical Factors Affecting Enzyme Activity

a. Temperature

• Effect: Reaction rate increases with temperature up to an optimal point, after which enzymes denature.
• Optimum: For most human enzymes, 35–40°C.

b. pH

• Effect: Deviations from the optimal pH alter enzyme structure and function.
• Examples:
  • Pepsin: Optimal pH ~2.
  • Trypsin: Optimal pH ~8.

c. Substrate Concentration

• Effect: Reaction rate increases with substrate concentration until saturation (when all active sites are occupied).
• Michaelis-Menten Behavior: Describes the relationship between substrate concentration and reaction velocity.

d. Enzyme Concentration

• Effect: Increasing enzyme concentration increases the reaction rate, provided substrate is not limiting.

e. Ionic Strength (Salt Concentration)

• Effect: Extreme salt concentrations disrupt ionic bonds, altering enzyme conformation and activity.

f. Presence of Inhibitors or Activators

• Effect:
  • Inhibitors: Reduce activity (competitive, non-competitive, irreversible).
  • Activators: Enhance activity (e.g., cofactors like Mg²⁺ or Zn²⁺).

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3. Regulation of Enzyme Activity

a. Allosteric Regulation

• Mechanism: Effector molecules bind to sites other than the active site, causing conformational changes.
• Types:
  • Allosteric Activation: Increases activity.
  • Allosteric Inhibition: Decreases activity.
• Example: ATP inhibits phosphofructokinase-1 (PFK-1) in glycolysis.

b. Feedback Inhibition

• Mechanism: End product of a pathway inhibits an upstream enzyme, preventing overproduction.
• Example: Isoleucine inhibits threonine deaminase.

c. Covalent Modification

• Mechanism: Reversible addition or removal of chemical groups (e.g., phosphorylation, acetylation).
• Examples:
  • Phosphorylation of glycogen phosphorylase activates it.
  • Dephosphorylation of enzymes by phosphatases.

d. Proteolytic Activation

• Mechanism: Inactive precursors (zymogens) are cleaved to form active enzymes.
• Examples:
  • Trypsinogen to trypsin in the digestive system.
  • Fibrinogen to fibrin in blood clotting.

e. Gene Expression Regulation

• Mechanism: Enzyme levels are controlled by transcriptional or translational changes.
• Example: Increased expression of lactase in response to lactose availability.

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Summary Table

Factor/Mechanism	Effect on Enzyme Activity	Example
Temperature	Optimal range enhances activity; extremes denature	Human enzymes: 35–40°C
pH	Optimal pH required for stability and activity	Pepsin (pH 2), Trypsin (pH 8)
Substrate Concentration	Activity increases until enzyme saturation	Michaelis-Menten kinetics
Allosteric Regulation	Activators/inhibitors modify activity	ATP inhibits PFK-1
Feedback Inhibition	End product inhibits upstream enzyme	Isoleucine inhibits threonine deaminase
Covalent Modification	Chemical changes modulate activity	Phosphorylation of glycogen phosphorylase
Proteolytic Activation	Zymogens activated by cleavage	Trypsinogen to trypsin