Lecture 7

Exam Review Overview

• Final Lecture Before the Exam: This lecture is crucial for consolidating knowledge before the exam on Monday.

• Review Session: A comprehensive review session is scheduled for Friday night at 3:30 PM in the usual lecture room. This is an excellent opportunity to clarify any doubts and reinforce learning before the final test.

• Exam Locations:

  • Students with last names A-L are to report to ILP.

  • Students with last names M-Z should go to LSV.

• Study Tips: It is recommended to utilize cheat sheets while studying to condense and organize information effectively.

Enzyme Mechanism: Trypsin

• Overview: Trypsin is a well-known digestive enzyme that plays a crucial role in breaking down proteins in the digestive system.

• Mechanism of Action: It speeds up the breakdown of proteins by cleaving peptide bonds specific to certain amino acid side chains, making it essential for protein digestion in the stomach.

• Active Site Characteristics: The active site of trypsin is uniquely shaped to accommodate the side chains of substrates (proteins), optimizing them for digestion.

• Denaturation of Substrates: During digestion, the initial substrate, often polypeptides, is typically denatured due to the acidic conditions prevailing in the stomach (pH ~1), which leads to their unfolding and makes the peptide bonds more accessible for cleavage.

Charge Relay System

• Mechanism: The serine oxygen in trypsin becomes highly reactive owing to a charge relay system. This system is significant for the catalytic mechanism and involves histidine as a key component.

• Catalytic Triad: The charge relay system comprises three critical components: serine, histidine, and aspartate, which together facilitate the enzymatic reaction.

• Substrate Binding: The substrate binds at the active site of the enzyme, triggering the subsequent reaction steps.

Reaction Mechanism Steps

1. Formation of the ES Complex: The initial step involves substrate binding to the enzyme's active site, which does not involve any chemical reaction at this stage.

2. Nucleophilic Attack: The process continues with the oxygen from the serine attempting to attack the carbon in the peptide bond, leading to the creation of an oxyanion intermediate—a temporary stable molecular structure.

3. Covalent Intermediate Formation: During the reaction, the enzyme-substrate complex is converted into a single molecule.

4. Cleavage of Peptide Bond: The peptide bond is cleaved, resulting in the formation of a new amino terminus, effectively releasing the product.

5. Regeneration of Enzyme: Finally, water is involved in hydrolysis, breaking the bond between the enzyme and the substrate and thus regenerating the active form of trypsin for further catalytic activity.

Key Points in Enzyme Function

• Catalytic Role: Enzymes function as biological catalysts, expediting reactions and returning to their original state post-reaction, which allows them to be reused in multiple catalytic cycles (recycling process).

• Acid-Base Catalysis: This plays a vital role in enzyme mechanisms, such as proton transfer between serine and histidine, which is essential for the enzymatic reaction process.

• Catalytic Efficiency: Enzymes exhibit remarkable catalytic efficiency, enabling them to perform numerous reactions without undergoing degradation.

Classification of Serine Proteases

• Specificity of Proteases: Serine proteases have different specificities determined by the shape and chemical properties of their active site pockets.

  • Trypsin: It shows specificity towards basic amino acids, including arginine and lysine.

  • Elastase: It accommodates smaller side chains such as glycine and alanine, showcasing the diversity in protease specificity.

• Binding Pocket vs. Catalytic Triad: The specificity is primarily defined by the characteristics of the binding pocket rather than the chemistry of the catalytic triad itself.

Enzyme Kinetics Overview

• Regulation Necessity: Enzymes require careful regulation to function optimally within biological systems.

• Michaelis-Menten Kinetics: This model elaborates on enzyme activity as it relates to substrate concentration, providing insights into enzyme efficiency.

  • Initial Velocity (V): Derived from the amount of substrate available and the rate constants (Km and Vmax), this parameter reflects how quickly a reaction proceeds.

Definitions

• Km (Michaelis Constant): This constant represents the substrate concentration at which the reaction velocity reaches half of Vmax, indicating the enzyme's affinity for its substrate. A low Km signifies high affinity, which means that the enzyme can effectively bind the substrate even at low concentrations.

• Vmax: It denotes the maximum rate achievable by the enzyme when it is fully saturated with substrate. Vmax reflects the total enzyme concentration and the turnover number, crucial for understanding enzyme efficiency.

Assumptions of Michaelis-Menten Kinetics

1. The reaction is conceptualized as a straightforward two-step process: E + S ⇌ ES → E + P.

2. Steady-State Assumption: It assumes that the concentration of the enzyme-substrate complex (ES) remains constant throughout the measurement period.

3. Rate-Limiting Step: The slowest step in the reaction sequence, involving the conversion of substrate into product, is regarded as the rate-limiting step influencing overall reaction speed.

Types of Inhibition

1. Competitive Inhibition:

   • The inhibitor competes with the substrate to bind at the active site of the enzyme.

   • This leads to an increase in Km (more substrate is needed to achieve half of Vmax) but does not alter Vmax because the inhibition can be overcome with higher substrate concentrations.

2. Noncompetitive Inhibition:

   • The inhibitor binds to an allosteric site, rendering the enzyme inactive regardless of substrate presence.

   • This results in a decrease in Vmax but does not affect Km, as the binding of the substrate is not impeded.

Enzyme Regulation and Activation

• Regulation Mechanisms: Enzymes can be regulated by various structural changes, which often involve:

  • Covalent Modifications: Such as phosphorylation or methylation, which can activate or inhibit enzyme function.

  • Proteolytic Cleavage: Activation of zymogens, protein precursors that require cleavage to become active enzymes. For example, trypsin is synthesized as an inactive zymogen and becomes active via cleavage.

  • Cascade Activation: Seen in processes like blood clotting, where enzymes activate one another in a series, amplifying responses during injury or other physiological changes.

Regulation of Enzyme Activity

• Enzymes need to be toggled on and off based on physiological requirements to maintain metabolic homeostasis. Proper regulation of enzymes is crucial in adapting to changes in cellular and overall body metabolism.

Additional Information for the Exam

• Review enzyme kinetics in detail, focusing on specific mechanisms and differentiation between various types of enzyme regulation and inhibition.

• Pay special attention to the functions and specificities of different proteases, particularly trypsin and elastase, and their practical implications in biological systems.

• Understand the significance of Km and Vmax in assessing enzyme efficiency and regulatory mechanisms, as these concepts are fundamental in enzyme kinetics and function evaluation.