Overview
This lecture covers enzyme catalysis, enzyme flexibility, mechanisms (with a focus on serine proteases), activation energy, Michaelis-Menten kinetics, allosteric enzymes, reaction mechanisms, and classification of enzymes.
Enzyme Catalysis & Flexibility
- Enzymes act as biological catalysts, greatly speeding up chemical reactions.
- Enzymes differ from chemical catalysts by being flexible and changing shape during reactions (induced fit model).
- The enzyme active site binds substrates, forming the enzyme-substrate (ES) complex.
- Substrate binding changes the enzyme’s shape, bringing reactants closer together.
- Enzymes return to their original state after the reaction, fulfilling the definition of a catalyst.
Activation Energy & Reaction Reversibility
- Activation energy (Ea) is the energy required to start a reaction.
- Enzymes lower activation energy but do not change overall free energy (ΔG) of the reaction.
- All reaction steps catalyzed by enzymes are reversible.
Serine Protease Mechanism
- Serine proteases cleave specific peptide bonds in proteins using an active site with a catalytic triad (serine, histidine, aspartic acid).
- Substrate binds in the S1 pocket, which provides specificity.
- The serine’s oxygen forms a reactive alkoxide ion, attacking the peptide bond.
- The oxyanion hole stabilizes reaction intermediates.
- The reaction proceeds in fast and slow steps, involving water and release of products.
Enzyme Kinetics & Michaelis-Menten Model
- Reaction velocity (Vâ‚€) is measured as product concentration over time.
- At low substrate, few enzymes are bound; at high substrate, enzymes are saturated (maximum velocity, Vmax).
- Initial velocity is measured to avoid reverse reaction effects.
- Steady state conditions occur when ES and free enzyme concentrations remain nearly constant.
- Michaelis-Menten equation: Vâ‚€ = (Vmax [S]) / (Km + [S]).
- Km reflects enzyme affinity for substrate (low Km = high affinity; high Km = low affinity).
- Vmax depends on enzyme quantity; kcat (turnover number) = Vmax / [enzyme], reflecting catalytic efficiency.
- kcat/Km ratio indicates enzyme “perfection” and efficiency.
Analyzing Enzyme Kinetics
- Lineweaver-Burk (double-reciprocal) plots help determine Vmax and Km precisely.
Allosteric Enzymes & Cooperativity
- Allosteric enzymes (often multi-subunit) show cooperative binding, producing a sigmoidal V vs. [S] curve.
- They exist in relaxed (R, active) or tight (T, inactive) states, explained by concerted or sequential models.
Enzyme Reaction Mechanisms
- Enzymes may follow random, ordered, or “ping-pong” (double displacement) substrate binding mechanisms.
- The ping-pong mechanism involves enzyme switching between covalently different states.
Enzyme Classification
- Enzymes are classified into six categories: oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases.
- Each type catalyzes a specific kind of reaction (e.g., transfer, hydrolysis, isomerization, joining, or breaking bonds).
Key Terms & Definitions
- Active Site — region on enzyme where substrate binds and reaction occurs
- Induced Fit Model — enzyme changes shape upon substrate binding
- Activation Energy (Ea) — minimum energy needed for a reaction to proceed
- Catalytic Triad — serine, histidine, aspartic acid residues critical for serine protease activity
- Oxyanion Hole — enzyme pocket stabilizing reaction intermediate
- Vmax — maximum reaction velocity at substrate saturation
- Km — substrate concentration at half Vmax; inverse measure of enzyme affinity
- kcat — turnover number, maximal number of substrate molecules converted per enzyme per second
- Steady State — condition where ES complex concentration is relatively constant
- Lineweaver-Burk Plot — double reciprocal plot to estimate Vmax and Km
Action Items / Next Steps
- Review Michaelis-Menten equation and practice plotting enzyme kinetics.
- Study the serine protease mechanism steps and identify the catalytic triad.
- Learn the six enzyme classification categories and examples of each.