03.12 Chapter Summary

Definition: Enzymes are globular proteins that act as biological catalysts.

Function: Speed up biochemical reactions without being consumed in the process.

Location:

• Intracellular Enzymes: Catalyze reactions inside cells.
• Extracellular Enzymes: Secreted outside cells to catalyze external reactions.

Active Site:

• Definition: Specific region on the enzyme where the substrate binds.
• Characteristics: Highly specific shape and chemical environment.

Enzyme–Substrate Complex:

• Formation: Substrate molecules bind to the enzyme’s active site, forming the complex.
• Stabilization: The complex stabilizes the transition state, reducing activation energy.

Lowering of Activation Energy:

• Mechanism: Enzymes lower the energy barrier required for reactions, increasing reaction rates.
• Outcome: More efficient and faster biochemical reactions.

Enzyme Specificity:

Lock-and-Key Hypothesis:

• Concept: Enzyme and substrate fit together perfectly without any alteration.
• Implication: High specificity; only specific substrates fit into specific enzymes.

Induced-Fit Hypothesis:

• Concept: Enzyme undergoes a conformational change to fit the substrate upon binding.
• Implication: Flexibility in enzyme structure enhances specificity and catalytic efficiency.

Measuring Reaction Rates:

Using Catalase:

• Function: Catalyzes the decomposition of hydrogen peroxide into water and oxygen.
• Measurement: Rate can be assessed by measuring the volume of oxygen gas produced over time.

Using Amylase:

• Function: Catalyzes the breakdown of starch into sugars.
• Measurement: Rate can be determined by measuring the decrease in starch concentration over time, often using iodine as an indicator.

Experimental Considerations:

• Control Variables: Temperature, pH, enzyme concentration, and substrate concentration should be controlled.
• Data Collection: Collect data at regular intervals to accurately determine reaction rates.

Colorimeter Basics:

• Function: Measures the absorbance of specific wavelengths of light by a solution.
• Application: Detects color changes associated with enzyme-catalyzed reactions.

Procedure:

• Prepare Samples: Set up reaction mixtures with the enzyme and substrate.
• Add Indicator: Use a color-changing reagent (e.g., iodine for starch with amylase).
• Measure Absorbance: At specific time intervals, use the colorimeter to measure absorbance changes.
• Interpret Results: Correlate absorbance changes to substrate concentration or product formation.

Advantages:

• Quantitative Data: Provides precise measurements of reaction progress.
• Real-Time Monitoring: Allows continuous tracking of reaction kinetics.

Introduction to Enzymes

• Enzymes are biological catalysts that speed up biochemical reactions without being consumed.
• Active Site: The specific region where substrates bind and reactions occur.
• Catalysis: Lowering the activation energy required for a reaction.

Factors Affecting the Rate of Enzyme-Catalysed Reactions

a. Temperature

• Effect:
  • Increasing Temperature: Generally increases reaction rate by providing more kinetic energy, leading to more frequent collisions between enzymes and substrates.
  • Optimal Temperature: Each enzyme has an optimal temperature at which its activity is highest.
  • Denaturation: Beyond the optimal temperature, enzymes denature (lose their structure) and become inactive.
• Graph: Rate vs. Temperature Optimal Point

b. pH (Using Buffer Solutions)

• Effect:
  • Optimal pH: Each enzyme operates best at a specific pH.
  • Deviation from Optimal pH: Alters the ionization of amino acids at the active site, affecting enzyme structure and function.
  • Buffer Solutions: Maintain a stable pH, ensuring consistent enzyme activity.
• Example:
  • Pepsin: Optimal pH ~2 (stomach).
  • Trypsin: Optimal pH ~8 (small intestine).

c. Enzyme Concentration

• Effect:
  • Increase in Enzyme Concentration: Higher enzyme levels increase the number of available active sites, boosting reaction rate.
  • Saturation Point: At high enzyme concentrations with limited substrate, the reaction rate continues to increase.

d. Substrate Concentration

• Effect:
  • Low Substrate Concentration: Rate increases linearly with substrate as more enzymes are occupied.
  • High Substrate Concentration: Enzymes become saturated; rate approaches a maximum (Vₘₐₓ) and levels off.
• Graph: Rate vs. Substrate Concentration

e. Inhibitor Concentration

Effect:

• Presence of Inhibitors: Decreases reaction rate by reducing effective enzyme concentration or altering active site.
• Competitive Inhibitors: Bind to active site, competing with substrate.
• Non-Competitive Inhibitors: Bind to allosteric site, altering enzyme structure and function.

a. Maximum Rate of Reaction (Vₘₐₓ)

• Definition: The highest possible rate of reaction when the enzyme is saturated with substrate.
• Significance: Represents the efficiency of the enzyme under optimal conditions.

b. Michaelis–Menten Constant (Kₘ)

• Definition: Substrate concentration at which the reaction rate is half of Vₘₐₓ.
• Derivation: Kₘ is derived from the Michaelis-Menten equation:

Affinity Indicator:

• Low Kₘ: High affinity (enzyme reaches Vₘₐₓ at low substrate concentrations).
• High Kₘ: Low affinity (requires higher substrate concentrations to reach Vₘₐₓ).
• Usage: Comparing Kₘ values helps determine which enzyme has a higher affinity for its substrate.

a. Competitive Inhibition

• Mechanism: Inhibitor resembles substrate and binds to the active site, preventing substrate binding.
• Effect on Kinetics:
  • Vₘₐₓ: Remains unchanged (can be reached by increasing substrate concentration).
  • Kₘ: Increases (higher substrate needed to achieve half Vₘₐₓ).
• Graphical Representation: Lineweaver-Burk plot shows lines intersecting on the Y-axis.

b. Non-Competitive Inhibition

• Mechanism: Inhibitor binds to an allosteric site, altering enzyme structure and function regardless of substrate presence.

Effect on Kinetics:

• Vₘₐₓ: Decreases (maximum rate cannot be achieved as some enzymes are always inhibited).
• Kₘ: Remains unchanged (affinity for substrate is unaffected).
• Graphical Representation: Lineweaver-Burk plot shows lines intersecting on the X-axis.

a. Activity: Immobilised vs. Free Enzymes

• Free Enzymes:
  • Mobility: Can freely diffuse and interact with substrates.
  • Stability: More susceptible to changes in environmental conditions (temperature, pH).
• Immobilised Enzymes:
  • Fixed Position: Bound to a solid support (e.g., alginate).
  • Stability: Enhanced resistance to denaturation and reuse in industrial processes.

b. Advantages of Using Immobilised Enzymes

• Reusability: Can be recovered and used multiple times, reducing costs.
• Stability: Increased thermal and pH stability extends enzyme lifespan.
• Ease of Separation: Simplifies product purification by easily separating enzymes from reaction mixtures.
• Controlled Environment: Immobilisation allows for better control over reaction conditions.

c. Example: Enzyme in Alginate

• Alginate Beads: Enzymes encapsulated in alginate gel retain activity while being immobilised.

Benefits:

• Protect enzymes from harsh conditions.
• Facilitate continuous processes in bioreactors.

a. Overview

• Fluid Mosaic Model: Describes the cell membrane as a dynamic and flexible structure with various proteins embedded in or associated with a fluid phospholipid bilayer.
• Dynamic Nature: Lipids and proteins can move laterally within the layer, allowing membrane flexibility and the formation of specialized regions.

b. Phospholipid Bilayer Formation

• Phospholipids: Amphipathic molecules with hydrophilic (water-attracting) heads and hydrophobic (water-repellent) tails.
• Hydrophobic Interactions: Tails face inward, shielded from water, while heads face the aqueous environment, creating a stable bilayer.
• Hydrophilic Interactions: Polar head groups interact with the surrounding water, maintaining membrane integrity.

c. Protein Arrangement

• Integral Proteins: Span the entire bilayer (transmembrane proteins), involved in transport and signaling.
• Peripheral Proteins: Attached to the membrane surface, involved in signaling and maintaining the cell’s shape.
• Protein Mobility: Proteins can move laterally, contributing to membrane fluidity and functionality.

a. Cholesterol

• Location: Interspersed within the phospholipid bilayer.
• Function: Modulates membrane fluidity, making it less fluid at high temperatures and preventing it from becoming too rigid at low temperatures.

b. Glycolipids

• Structure: Lipids with carbohydrate chains attached.
• Location: Outer leaflet of the cell membrane.
• Function: Involved in cell recognition and protection.

c. Glycoproteins

• Function: Play roles in cell recognition, signaling, and adhesion.
• Structure: Proteins with carbohydrate chains attached.
• Location: Outer surface of the cell membrane.