General Biology I - Lecture 3: Enzymes

Introduction to Enzymes

• Enzymes are globular proteins consisting of long chains of amino acids folded into specific three-dimensional shapes, acting as biological catalysts.
• They speed up or slow down metabolic reactions without being permanently altered or consumed in the process.
• Enzymes facilitate:
  • The breaking of existing covalent bonds.
  • The formation of new chemical bonds.
• They provide a pathway with a lower activation energy but do not change the net free energy ($\Delta G$) or the equilibrium of a reaction.

Key Concepts

1. Substrates and Products

• Substrates: The specific reactant molecules that bind to the enzyme.
• Products: The resulting substances released from the active site after the reaction reaches completion.

2. Active Sites and Transition States

• Active Site: A small, three-dimensional pocket or cleft within an enzyme where substrate molecules bind.
  • Forms an enzyme-substrate (ES) complex.
  • The $R$ groups (side chains) of amino acids at the active site form temporary bonds (hydrogen bonds, ionic interactions, or hydrophobic interactions) with the substrate.
  • These interactions stabilize the transition state, a high-energy intermediate state where bonds are being formed or broken.

3. Activation Energy

• Activation Energy ($E_a$): The minimum energy required for substrates to reach the transition state and convert into products.
• Enzymes reduce $E_a$ by:
  • Distorting the substrate's shape to weaken existing bonds.
  • Providing a microenvironment (e.g., specific pH) conducive to the reaction.
  • Bringing multiple substrates together in the correct orientation.

4. Enzyme Specificity

Lock and Key Hypothesis (Emil Fischer, $1894$)

• Proposes that the active site and substrate shapes are complementary and fit together perfectly.
• Limitation: It does not account for the dynamic flexibility observed in proteins during catalysis.

Induced Fit Hypothesis (Daniel Koshland, $1958$)

• Suggests that the active site is not a perfect rigid match initially.
• As the substrate approaches, the active site reshapes its conformation due to chemical interactions, creating a snug fit.
• This strain on the enzyme-substrate complex helps lower the activation energy.

Enzyme-Catalyzed Reactions

Measurement Methods

1. Formation of $O_2$ from Catalase

• Reaction: $2H<em>2O</em>2 \xrightarrow{\text{Catalase}} 2H<em>2O + O</em>2$
• Procedure:
  • Mash biological material (e.g., potato tuber or celery) and filter to obtain a catalase solution.
  • Combine with hydrogen peroxide ($H<em>2O</em>2$) in a test tube.
  • Collect the oxygen gas produced using a gas syringe or by water displacement, tracking volume over time ($t$) to determine the initial rate.

2. Disappearance of Starch by Amylase

• Procedure:
  • Mix amylase solution with a starch suspension.
  • Periodically take samples to test for starch using iodine in $KI$ solution:
  • Iodine turns dark blue/black in the presence of starch.
  • The color remains orange-brown (iodine's natural color) once all starch is hydrolyzed into maltose.
  • A colorimeter can be used to measure the change in light absorbance quantitatively.

Factors Affecting Enzyme Activity

Key Factors

1. Temperature

   • Increasing temperature increases the kinetic energy ($KE$) of molecules, raising the frequency of successful collisions.
   • Optimal Temperature: The temperature at which the rate of reaction is highest ($\approx 37^{\circ}C$ for humans).
   • Denaturation: At high temperatures (usually above $45-50^{\circ}C$), the increased vibration breaks weak hydrogen and ionic bonds, causing the enzyme to lose its tertiary structure and active site shape.

2. $pH$

   • Changes in $[H^+]$ concentration affect the ionization of $R$ groups at the active site.
   • Extreme $pH$ levels lead to denaturation by disrupting ionic bonds.
   • Examples:
     • Pepsin (Stomach): Optimal $pH \approx 1.5$.
     • Trypsin (Small Intestine): Optimal $pH \approx 8.0$.

3. Enzyme and Substrate Concentration

   • Enzyme Concentration: Rate is directly proportional to enzyme concentration as long as substrate is in excess ($v \propto [E]$).
   • Substrate Concentration: Rate increases with $[S]$ until all active sites are occupied (saturation).

4. Inhibitors

   • Competitive Inhibitors: Bind to the active site; can be overcome by increasing $[S]$. They increase $K<em>m$ but do not change $V</em>{max}$.
   • Non-competitive Inhibitors: Bind to an allosteric site, changing the enzyme's shape. They decrease $V<em>{max}$ but do not change $K</em>m$.

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