5.2 - Enzymes

Definition and Necessity of Enzymes

• Biochemical reactions must occur rapidly in cells to maintain living processes.
• Enzymes accelerate these biochemical reactions.
• Enzyme: An organic catalyst, mostly made up of proteins, produced by cells of living organisms.
• Not all enzymes are synthesised from proteins.
• Substrate: A substance required for an enzyme reaction.
• A substrate binds to an enzyme at a specific active site to form an enzyme-substrate complex.

Enzyme Nomenclature

• In the 1960s, the International Union of Biochemistry and Molecular Biology (IUBMB) introduced enzyme nomenclature based on the substrate or reaction catalysed.
• The enzyme name is derived by adding -ase to the name of its substrate.
  • Example: Lactase catalyses the hydrolysis of lactose.
  • $\text{Lactose}+\text{water}\stackrel{\text{Lactase}}{\to }\text{Glucose}+\text{galactose}$
• Enzymes discovered before systematic nomenclature may retain conventional names, such as trypsin, pepsin and renin.

General Characteristics of Enzymes

• Biological catalysts that accelerate biochemical reactions.
• Act rapidly.
• Required in small quantities and reusable.
• Remain unchanged and are not destroyed after a reaction.
• Most enzyme-catalysed reactions are reversible.
• Specific: Only a substrate with a complementary shape can bind to the active site.
• Some require cofactors, such as vitamin B and magnesium ions, to work more efficiently.
• Inhibitors can slow or stop enzyme activity; examples include lead and mercury.

Intracellular and Extracellular Enzymes

• Intracellular enzymes: Synthesised in a cell for its own use.
  • Example: Hexokinase is used in glycolysis during cellular respiration.
• Extracellular enzymes: Secreted outside the cell.
  • Example: Trypsin is produced by pancreatic cells and secreted into the duodenum to break down polypeptides.

Production of Extracellular Enzymes

1. Ribosome is the site of protein synthesis.
2. Proteins synthesised by ribosomes enter the lumen of the rough endoplasmic reticulum and are transported through it.
3. At the end of the rough endoplasmic reticulum, the membrane buds off to form transport vesicles.
4. Transport vesicles containing proteins move to and fuse with the Golgi apparatus.
5. The Golgi apparatus modifies the proteins into enzymes and packages them in secretory vesicles formed from its tip.
6. Secretory vesicles move to and fuse with the plasma membrane to secrete extracellular enzymes.

Mechanism of Enzyme Action

The ‘Lock and Key’ Hypothesis

• Most enzymes are complex proteins with polypeptide chains folded into three-dimensional structures.
• The active site has a specific configuration complementary to a specific substrate.
• The enzyme represents the lock; the substrate represents the key.
• Sequence:
  1. The specific substrate approaches the enzyme.
  2. The substrate binds to the active site, forming an enzyme-substrate complex.
  3. A reaction occurs; products form and leave the active site.

Activation Energy

• Activation energy: Energy needed to break bonds in substrate molecules before a reaction can occur.
• Most reactions in cells require high activation energy.
• Enzymes lower activation energy and accelerate biochemical reactions in cells.

Factors Affecting the Mechanism of Enzyme Action

• Temperature, pH, substrate concentration and enzyme concentration affect enzyme action.

Effect of Temperature

1. Low temperature:
   • Enzyme-catalysed reaction rate is low.
2. Temperature increases:
   • Kinetic energy of enzyme and substrate molecules increases.
   • Effective collision frequency increases.
   • Reaction rate increases and doubles for every $10^{\circ }\text{C}$ rise until the optimum temperature.
3. Optimum temperature:
   • Reaction rate is maximum.
   • Optimum temperature for enzymes in the human body is about $37^{\circ }\text{C}$.
4. Above the optimum temperature:
   • Activity decreases rapidly and stops at $60^{\circ }\text{C}$.
   • High temperature breaks chemical bonds in the enzyme; the enzyme is denatured.
   • The three-dimensional structure and active site change, so the substrate is no longer complementary to the active site.

Effect of pH

• Enzymes act most effectively at their optimum pH.
• Most enzymes are most active between pH 6 and 8.
  • Salivary amylase: pH 6.8.
  • Pepsin in the stomach: pH 1.5–2.5.
  • Trypsin in the duodenum: About pH 8.5.
• A pH change alters the charge of the active site and substrate surface; the enzyme-substrate complex cannot form.
• If pH returns to optimum, the active-site charge is restored and the enzyme functions normally.
• An extreme pH change breaks structural chemical bonds, changes the active site and denatures the enzyme.

Effect of Substrate Concentration

• Conditions: Enzyme concentration is fixed; substrate concentration increases.
• More substrate increases effective collisions and product formation.
• Reaction rate increases until it reaches a maximum, then remains constant.
• At maximum rate:
  • all active sites are saturated with substrate;
  • enzyme concentration is the limiting factor;
  • adding more enzyme increases the reaction rate.

Effect of Enzyme Concentration

• More enzyme provides more active sites and increases reaction rate.
• Doubling enzyme concentration doubles the substrate converted into products per unit time if substrate is in excess.
• At maximum rate:
  • substrate concentration is the limiting factor;
  • adding more substrate increases the reaction rate.

Experiments on Enzyme Activity

Effect of Temperature on Amylase Activity

• Problem statement: What is the effect of temperature on the reaction rate of amylase?
• Hypothesis: Reaction rate increases with temperature up to the optimum temperature, then decreases.
• Variables:
  • Manipulated: Temperature.
  • Responding: Reaction rate of amylase.
  • Fixed: Concentrations of amylase and starch suspension; pH of the reaction medium.
• Method:
  1. Incubate separate 1% starch suspensions and 0.5% amylase solutions for 5 minutes in water baths at $20^{\circ }\text{C}$, $30^{\circ }\text{C}$, $40^{\circ }\text{C}$, $50^{\circ }\text{C}$ and $60^{\circ }\text{C}$.
  2. Mix each temperature-matched pair and start the stopwatch immediately.
  3. Test samples with iodine solution at fixed intervals while keeping the mixtures in their water baths.
  4. Record the time when starch hydrolysis is complete: The iodine mixture remains brownish yellow.
  5. Calculate $\text{reaction rate}=1÷\text{time}$ and plot reaction rate against temperature.

Effect of pH on Pepsin Activity

• Problem statement: What is the optimum pH for a pepsin reaction?
• Hypothesis: pH 2 is optimum for the pepsin reaction.
• Variables:
  • Manipulated: pH of the reaction medium.
  • Responding: Clarity or turbidity of the reaction mixture.
  • Fixed: Concentrations of albumen and pepsin solutions; temperature of the reaction medium.
• Method:
  1. Prepare three test tubes containing 5 ml albumen suspension.
  2. Add 1 ml of 1% pepsin solution and, respectively, 1 ml of 0.1 M hydrochloric acid, distilled water or 0.1 M sodium hydroxide solution.
  3. Measure and record each mixture’s pH.
  4. Incubate all tubes at $37^{\circ }\text{C}$ for 20 minutes.
  5. Record clarity or turbidity at 0 minutes and after 20 minutes.