11-Lecture_11_3

Molecules to Cells, Part 1: Enzymes

• Presenter: Prabodhika Mallikaratchy, Ph.D.

• Date: January 17, 2024

Learning Objectives

• Define enzymes and describe their fundamental structural and thermodynamic properties.

• Recognize different classes of enzymes and their associated reactions.

• Describe representative mechanistic strategies and mechanisms of enzyme catalysis.

Enzymes Overview

• Enzymes are catalysts that increase reaction rates without being consumed in the process.

• Many enzymes have a globular (soluble in water) protein structure.

• They typically operate under life-compatible conditions: 37°C and a pH of 7.2-7.4.

Enzymatic Reactions: Rate and Measurements

• Substrate (S): starting material in enzymatic reactions (e.g., Glyceraldehyde-3-phosphate).

• Product (P): result of the reaction (e.g., Dihydroxyacetone phosphate).

• Enzymes are represented by E (e.g., Triose phosphate isomerase).

• Reaction progress: substrate concentration [S] decreases over time, while product concentration [P] increases.

• Velocity (v) can be expressed as:

  • v = - (d[S]/dt) or v = (d[P]/dt).

Enzyme Active Site

• Each enzyme has a specific active site designed to bind its substrate.

• Active sites are clefts or pockets formed by the folding of the protein.

• Enzyme specificity is determined by the arrangement of amino acids in the active site.

• Common features of active sites:

  • Three-dimensional structure with a unique crevice for substrate binding.

  • Creation of a microenvironment favorable for the reaction.

  • Binding involves multiple weak interactions with the substrate.

Models of Enzyme-Substrate Interaction

• Lock and Key Model: Substrate fits perfectly into the enzyme's active site.

• Induced Fit Model: Enzyme adapts its shape to accommodate an ill-fitting substrate, facilitating more interactions.

Thermodynamic Properties of Enzymes

• Transition state (TS) is the point during a reaction where reactants are transformed into products.

• Energy diagrams illustrate the relationship between substrate (S), transition state (TS), and products (P).

• Activation energy (Ea) is the energy required to reach the transition state.

• Enzymes lower the activation energy, speeding up the reaction without altering the total energy change (ΔG) from substrates to products.

Enzyme Function Mechanisms

• Various strategies enzymes use to catalyze reactions:

  • Acid-base catalysis: proton transfer aids reaction.

  • Covalent catalysis: formation of temporary covalent bonds.

  • Metal ion catalysis: metal ions facilitate reactions.

  • Catalysis by approximation: reactive groups are brought together efficiently.

Classes of Enzymes

Six Major Classes:

1. Oxidoreductases: Catalyze oxidation/reduction reactions (e.g., lactate dehydrogenase).

   • Involves electron and hydrogen transfer.

2. Transferases: Transfer functional groups (e.g., serine hydroxy-methyl transferase).

3. Hydrolases: Cleavage of bonds through the addition of water (e.g., urease).

4. Lyases: Catalyze addition/removal of groups to form double bonds (e.g., pyruvate decarboxylase).

5. Isomerases: Catalyze isomerization reactions (e.g., triose phosphate isomerase).

6. Ligases: Join two substrates using ATP (e.g., pyruvate carboxylase).

Enzyme Catalyzed Reactions

• Proteases: Enzymes that cleave peptide bonds.

  • Chymotrypsin is a serine protease involved in protein digestion and is activated from zymogen forms.

  • It selectively targets hydrophobic amino acids.

• Chymotrypsin Mechanism: Consists of several steps, including substrate binding, nucleophilic attack, and product dissociation involving a catalytic triad (Ser195, His57, Asp102).

• Enzyme Specificity: Variations in binding pockets allow different substrate selectivity among enzymes like chymotrypsin, trypsin, and elastase.

Chymotrypsin Mechanism

Chymotrypsin is a serine protease that plays an essential role in the digestion of dietary proteins. It is synthesized as an inactive precursor, known as a zymogen (specifically, trypsinogen), and is activated in the gastrointestinal tract. The mechanism of chymotrypsin's action involves several critical steps:

1. Substrate Binding: The substrate, a protein or peptide, enters the active site of chymotrypsin. The specificity of chymotrypsin allows it to preferentially bind to hydrophobic amino acid residues, due to the specific structure of its active site.

2. Nucleophilic Attack: The catalytic triad, consisting of Serine 195 (Ser195), Histidine 57 (His57), and Aspartate 102 (Asp102), plays a key role. When the substrate is bound, His57 acts as a base, accepting a proton from Ser195, which increases the nucleophilicity of the serine's hydroxyl group. This makes Ser195 ready to attack the carbonyl carbon of the peptide bond in the substrate.

3. Formation of a Tetrahedral Intermediate: The nucleophilic attack by Ser195 results in the formation of a tetrahedral intermediate, destabilizing the peptide bond and leading to the formation of a covalent enzyme-substrate complex.

4. Decomposition of Tetrahedral Intermediate: This intermediate collapses, breaking the peptide bond and releasing the C-terminal portion of the substrate as the first product. His57 donates a proton to the amino group of the cleaved substrate, facilitating its dissociation.

5. Water Molecule Activation: A water molecule enters the active site and is activated similarly to the way Ser195 was activated earlier. His57 works as a base once again, abstracting a proton from the water molecule, allowing it to act as a nucleophile.

6. Second Nucleophilic Attack: The activated water molecule attacks the acyl-enzyme intermediate (the enzyme-substrate complex), forming a second tetrahedral intermediate.

7. Completion of Reaction: The collapse of this second intermediate leads to the release of the second product, which is the original N-terminal portion of the substrate. The enzyme is regenerated in its active form, ready for another catalytic cycle.

Importance of Chymotrypsin

Chymotrypsin not only illustrates enzyme catalysis principles but also serves a specific physiological function in protein digestion. Its selective cleavage pattern contributes to various metabolic pathways in cells, highlighting the enzyme's role beyond mere digestion. Additionally, understanding its mechanism offers insights into the design of protease inhibitors used in pharmaceutical applications, particularly in treating conditions like chronic pancreatitis and certain cancers.