Chapter 6: Enzymes – The Catalysts of Life (Vocabulary Flashcards)

Activation energy and the role of enzymes

• Nearly all cell reactions require a catalyst; these biological catalysts are called enzymes. They work by lowering the activation energy of a reaction.

• Activation energy is the minimum energy required to start a chemical reaction. Enzymes lower this barrier, allowing more molecules to reach the transition state and form products.

• Transition state (brief): the point in a reaction where reactants have their highest energy and are on the verge of being converted into products.

Mechanism and enzyme structure

• Enzymes are primarily proteins.

• Substrates bind to enzymes at a specific region called the active site, forming the enzyme–substrate complex.

• The active site shape and chemistry facilitate the chemical transformation of the substrate to products.

• Some enzymes require cofactors to function; cofactors can be inorganic or organic. Organic cofactors are called coenzymes and are usually derived from vitamins or minerals.

Enzyme classes (six major classes)

• Oxidoreductases: catalyze oxidation–reduction (electron transfer) reactions. Example: Lactate dehydrogenase.

• Transferases: transfer functional groups between molecules. Example: Kinase.

• Hydrolases: break bonds via hydrolysis using water. Example: Lipase.

• Lyases: break bonds without water or redox changes; often form double bonds or rings. Example: Decarboxylase.

• Isomerases: rearrange atoms within a molecule (isomerization). Example: Phosphoglucose isomerase.

• Ligases: join two molecules together, often using ATP. Example: DNA ligase.

Enzyme sensitivity and environmental factors

• Temperature: Too high temperatures can cause denaturation; temperatures that are too low slow or stop enzymatic activity.

• pH: Changes in pH alter the charges of amino acids at the active site, influencing substrate binding and enzyme activity.

Inhibitors and activators; basic model of enzyme activity

• An enzyme can be inhibited or activated by various molecules.

• Inhibitors can prevent substrate binding or disrupt catalysis; activators enhance activity.

• A general schematic: substrate binds, reaction proceeds, product is formed; an inhibitor can bind to the active site and prevent the substrate from binding, inhibiting the reaction.

Models of substrate interaction with enzymes

• Lock & Key model: the active site is a perfect fit for the substrate; no conformational change is required.

• Induced fit model: the enzyme's active site changes shape slightly to accommodate the substrate when binding occurs.

• Visuals:

  • Lock & Key: substrate sits in the fixed active site.

  • Induced Fit: active site adapts to snugly accommodate the substrate, enhancing catalysis.

Michaelis–Menton kinetics: historical context and key concepts

• Michaelis & Menton (historical contributors) developed a mathematical model describing how the reaction rate depends on substrate concentration.

• Observations: as substrate concentration increases, the reaction rate increases until it reaches a maximum speed (Vmax) that cannot be exceeded even with more substrate; this occurs when all enzyme active sites are saturated.

• Initial velocity concept: at low substrate concentrations, many enzymes are free to bind substrate and increase the rate; as [S] grows, enzymes become saturated and the rate levels off.

Key quantities in Michaelis–Menten kinetics

• Vmax: the maximum rate of an enzyme-catalyzed reaction, achieved when all enzyme active sites are saturated with substrate.

• Km (Michaelis constant): the substrate concentration at which the reaction rate is half of Vmax.

• Relationships:

  • At low [S], the rate increases nearly linearly with [S].

  • At high [S], the rate approaches Vmax as enzymes become saturated.

• Definitions in formulas:

  • Michaelis–Menten equation: $v = rac{V<em>{ ext{max}} [S]}{K</em>m + [S]}.$

  • At half-maximum velocity: when $v = rac{V<em>{ ext{max}}}{2}$, then $[S] = K</em>m$.

Practical notes on Vmax and Km estimation

• Vmax is the plateau of the Michaelis–Menten curve; it can be challenging to estimate precisely because infinite substrate concentration is not practical.

• Km provides a measure of substrate affinity: a lower Km indicates higher affinity; a higher Km indicates lower affinity.

• Converting experimental data to LMN plots (Lineweaver–Burk) can help estimate these parameters, though it can introduce biases.

Example data and estimation contrasts (from the transcript)

• Michaelis–Menten method (approximate):

  • Vmax ≈ 7.75

  • Km ≈ 0.75

• Lineweaver–Burk plot method (in the transcript):

  • Vmax ≈ 10

  • Km ≈ 0.15

• Note: These two methods can yield different estimates; the Lineweaver–Burk plot exaggerates error at low substrate concentrations.

• Practical reminders:

  • Units of Vmax: concentration per unit time (e.g., $ext{M}\, ext{s}^{-1}$ or $ext{µM}\, ext{min}^{-1}$).

  • Units of Km: concentration (e.g., $ext{M}$ or $ext{µM}$).

Lineweaver–Burk plot: double reciprocal plot

• Transformation: invert the Michaelis–Menten equation to linear form.

• Equation: $rac{1}{v} = rac{K<em>m}{V</em>{ ext{max}}} rac{1}{[S]} + rac{1}{V_{ ext{max}}}.$

• Plot: y-axis = $rac{1}{v}$, x-axis = $rac{1}{[S]}$.

• Linear characteristics:

  • Slope = $rac{K<em>m}{V</em>{ ext{max}}}$

  • Y-intercept = $rac{1}{V_{ ext{max}}}$

  • X-intercept = $- rac{1}{K_m}$

• Usage: helps visualize changes in Vmax and Km under different conditions or with inhibitors.

Turnover number (kcat)

• Definition: Turnover number is the number of substrate molecules converted to product per enzyme molecule per unit time when the enzyme is fully saturated.

• Formula: $k<em>{ ext{cat}} = rac{V</em>{ ext{max}}}{[E]}<br>$ where $[E]$ is the total enzyme concentration (active sites available).

• Example 1 (from the transcript):

  • If the enzyme concentration is [E] = 5 imes 10^{-5}\n ^{-6} ext{ M} (i.e., 5x10^{-5} µM) and $V<em>{ ext{max}} = 3 ext{ µM/min}$, then $k</em>{ ext{cat}} = rac{3}{5 imes 10^{-5}} = 6.0 imes 10^{4} ext{ min}^{-1}.$

  • Interpretation: each enzyme molecule can convert about 60,000 substrate molecules per minute when saturated.

• Example 2 (from the transcript):

  • If an $ext{enzyme}$ solution of $[E] = 0.004 ext{ M}$ produces 0.32 product in 1 s, then
    $k_{ ext{cat}} = rac{0.32}{0.004} = 80 ext{ s}^{-1}.$

Enzyme inhibition and regulation (reversible and irreversible)

• Irreversible inhibitors: molecules that permanently bind to an enzyme (often at the active site) and inactivate it.

• Reversible inhibitors: bind temporarily; enzyme activity can be restored when the inhibitor dissociates.

• Competitive inhibition (reversible): inhibitor binds to the active site, blocking substrate binding.

  • Effect: Vmax remains the same; Km increases (apparent affinity decreases).

• Noncompetitive inhibition (reversible): inhibitor binds to a site other than the active site, altering enzyme function.

  • Effect: Km remains the same; Vmax decreases.

• Uncompetitive inhibition (reversible): inhibitor binds only to the enzyme-substrate complex.

  • In the transcript, it is stated as Km increases and Vmax decreases; standard biochemistry typically shows both Km and Vmax decrease.

• Mixed inhibition (reversible): inhibitor can bind to either the free enzyme or the enzyme–substrate complex; results in different effects depending on binding.

• Pure non-competitive inhibition: a type where Vmax decreases while Km remains unchanged (special case of noncompetitive).

Allosteric regulation and other control mechanisms

• Allosteric enzymes: regulated by molecules binding at allosteric sites (sites other than the active site); binding changes the enzyme’s shape and activity.

• Feedback inhibition: in multistep pathways, the end product slows down or stops the entire process by inhibiting an early enzyme.

• Covalent modification: activity is regulated by adding or removing chemical groups (e.g., phosphate or methyl groups) to modify enzyme function.

• Proteolytic cleavage: activation or inactivation of a protein by cutting its peptide chain (e.g., zymogen activation).

Real-world example: pepsin and the gastric system

• Pepsin is formed by proteolytic cleavage of pepsinogen (a zymogen) to become an active enzyme.

• Cells involved: Parietal cells secrete HCl; Chief cells secrete pepsinogen; gastric glands contain both.

• Dietary proteins are partially digested by pepsin in the stomach.

Practical notes and key takeaways

• Enzymes dramatically accelerate reactions by lowering the activation energy, not by changing the equilibrium position.

• The active site’s shape and chemistry, plus potential cofactors/coenzymes, determine substrate binding and catalysis.

• Temperature and pH must be within appropriate ranges for maximal activity; deviations can denature or inhibit enzymes.

• Kinetic parameters Vmax and Km summarize catalytic efficiency under specific conditions; they can be experimentally estimated by Michaelis–Menten fits or Lineweaver–Burk plots, each with pros/cons.

• The Lineweaver–Burk plot linearizes the MM equation for better visual comparison of enzyme behavior under different conditions or inhibitors, though it can overweight data at low substrate levels.

• Turnover number kcat provides a per-enzyme-molecule rate under saturating substrate conditions and helps compare catalytic efficiencies across enzymes.

• Inhibitors can modulate activity via competitive, noncompetitive, uncompetitive, or mixed mechanisms, with characteristic effects on Vmax and Km; allosteric regulation and covalent modification add further layers of control.

• The study of enzyme kinetics connects to real-world biology: digestion (pepsin), metabolism, drug design, and regulation of metabolic pathways.

$v = rac{V<em>{ ext{max}} [S]}{K</em>m + [S]}$

$rac{1}{v} = rac{K<em>m}{V</em>{ ext{max}}} rac{1}{[S]} + rac{1}{V_{ ext{max}}}$

$k<em>{ ext{cat}} = rac{V</em>{ ext{max}}}{[E]}$

Km definitions and relationships:

• At half-maximum velocity: $[S] = K<em>m$ when $v = frac{V</em>{ ext{max}}}{2}$.

Notes on units:

• Vmax units: concentration per time, e.g., $ext{M s}^{-1}$ or $ext{µM min}^{-1}$.

• Km units: concentration, e.g., $ext{M}$ or $ext{µM}$.

Example calculations (referenced):

• Example 1: $k<em>{ ext{cat}} = rac{V</em>{ ext{max}}}{[E]} = rac{3}{5 imes 10^{-5}} = 6.0 imes 10^{4} ext{ min}^{-1}$.

• Example 2: If $[E] = 0.004 ext{ M}$ and product = 0.32 in 1 s, then
  $k_{ ext{cat}} = rac{0.32}{0.004} = 80 ext{ s}^{-1}$.