Enzymes, Structure & Action

Enzymes are biological catalysts, proteins that speed up chemical reactions in living organisms without being consumed in the process. Virtually every metabolic reaction in cells requires an enzyme to proceed at a biologically useful rate.

Key Properties of Enzymes

Enzymes are globular proteins with a specific three-dimensional shape that is essential for their function. This shape is maintained by various bonds and interactions within the protein molecule.

Levels of Protein Structure in Enzymes

The Active Site

The active site is a specific region on the enzyme surface where substrate molecules bind and chemical transformation occurs. Key features include:

• Specific pocket or cleft formed by the folding of the polypeptide chain
• Complementary shape to the substrate molecule(s)
• Specific amino acid residues that interact chemically with the substrate
• Usually relatively small compared to the overall enzyme size (typically 3-12 amino acids)

Cofactors and Coenzymes

Many enzymes require additional non-protein components to function:

Two major models describe how enzymes interact with their substrates: the lock-and-key model and the induced fit model.

Enzyme-catalysed reactions follow a consistent cycle that can be summarised in several key steps:

1. Substrate binding: The substrate molecule(s) collide with the enzyme and bind to the active site, forming an enzyme-substrate (ES) complex. This involves weak interactions such as hydrogen bonds and ionic bonds.
2. Induced fit: The enzyme undergoes a conformational change that brings catalytic residues into optimal position and may place strain on substrate bonds.
3. Catalysis: The reaction occurs at the active site. The enzyme may facilitate the reaction by: (a) orienting substrates correctly, (b) straining substrate bonds, (c) providing a favourable microenvironment, or (d) participating directly in the reaction.
4. Product formation: The reaction reaches completion, converting substrate to product(s) within the enzyme-product (EP) complex.
5. Product release: The product(s) have lower affinity for the active site and are released, freeing the enzyme to bind another substrate molecule.

Activation Energy and the Transition State

Enzymes accelerate reactions by lowering the activation energy (Eₐ), the energy barrier that must be overcome for a reaction to proceed. They do this by:

• Stabilising the transition state (the high-energy intermediate configuration)
• Providing an alternative reaction pathway with lower Eₐ
• Bringing reactants into close proximity and correct orientation

Several environmental factors influence enzyme activity. Understanding these is crucial for both biological systems and industrial applications.

1. Temperature

• As temperature increases, kinetic energy increases, leading to more frequent enzyme-substrate collisions
• Rate approximately doubles for every 10°C rise (Q₁₀ ≈ 2), up to an optimum
• Optimum temperature: Usually around 37°C for human enzymes (body temperature)
• Above optimum: increased vibration disrupts weak bonds holding the tertiary structure; enzyme denatures
• Thermophilic organisms have enzymes with higher optimum temperatures (e.g., 70-80°C)

2. pH

• Affects ionisation of amino acid residues in the active site
• Each enzyme has an optimum pH where activity is maximal
• Most human enzymes: optimum pH ~7 (neutral), e.g., amylase in saliva
• Pepsin (stomach): optimum pH ~2 (acidic environment)
• Trypsin (small intestine): optimum pH ~8 (alkaline environment)
• Extreme pH disrupts ionic bonds, causing denaturation

3. Substrate Concentration

• At low [S], reaction rate is proportional to substrate concentration
• As [S] increases, rate increases but progressively less
• At high [S], rate plateaus, enzyme becomes saturated; all active sites occupied
• Maximum rate = Vₘₐₓ; at half Vₘₐₓ, [S] = Kₘ (Michaelis constant, a measure of enzyme affinity)

4. Enzyme Concentration

• Directly proportional to reaction rate (when substrate is not limiting)
• More enzyme molecules = more active sites available

5. Inhibitors

Type	Mechanism	Effect	Example
Competitive	Reversible binding to active site	Competes with substrate; increased [S] can overcome	Malonate (inhibits succinate dehydrogenase)
Non-competitive	Binding to allosteric site	Changes enzyme shape; cannot be overcome by [S]	Cyanide (inhibits cytochrome oxidase)

Denaturation is the irreversible loss of an enzyme's three-dimensional structure, resulting in loss of biological activity. Unlike inhibitors which temporarily block function, denaturation permanently destroys the active site.

Mechanism of Denaturation

1. Weak bonds maintaining tertiary structure are disrupted
2. Protein unfolds from its globular shape
3. The specific 3D shape of the active site is lost
4. Substrate can no longer bind effectively
5. Catalytic activity is abolished

Biological and Practical Significance

Understanding enzyme structure and function has led to numerous practical applications in medicine and industry.

Medical Applications

Industrial Applications

This practical investigation explores how pH affects the activity of catalase, an enzyme that breaks down hydrogen peroxide into water and oxygen.

Background

Catalase is found in many living tissues, including liver and potato. It catalyses the decomposition of hydrogen peroxide (H₂O₂), a toxic byproduct of metabolism:

2H₂O₂ → 2H₂O + O₂

Materials

• Fresh liver homogenate or potato extract (catalase source)
• Hydrogen peroxide solution (3%)
• Buffer solutions at pH 3, 5, 7, 9, 11
• Test tubes, measuring cylinders, stopwatch
• Gas syringe or inverted measuring cylinder for O₂ collection

Method

1. Add 5 mL of buffer solution to each of 5 test tubes (one for each pH)
2. Add 2 mL of liver homogenate to each tube and mix
3. Add 5 mL of H₂O₂ to each tube and immediately start timing
4. Measure oxygen production (volume of foam or gas collected) at 30-second intervals for 3 minutes
5. Record results in a table

Results Table

Discussion Questions

1. At which pH was enzyme activity highest? Why?
2. What happens to the enzyme at very low or very high pH?
3. Why is it important to use buffer solutions rather than simply adding acid or base?
4. How could you improve the reliability of your results?
5. What is the biological significance of catalase having an optimum pH around 7?

Enzyme-substrate binding happens at a molecular scale that cannot be observed directly with school equipment. To meet the syllabus requirement to model the action of enzymes in cells, this investigation uses simple physical models to compare the lock-and-key and induced fit mechanisms described earlier in this lesson.

Aim

To construct physical models of the lock-and-key and induced fit mechanisms of enzyme action, and to evaluate which model better represents real enzyme behaviour.

Materials

• Card or foam board, cut into one fixed "active site" shape and several matching and non-matching "substrate" shapes
• Modelling clay or plasticine, moulded into a second "active site"
• A range of small objects of different shapes and sizes to act as substrates (e.g. blocks, marbles, irregular pieces)
• Ruler and stopwatch

Method

1. Lock-and-key model: Cut a rigid card "active site" with one exact complementary shape. Time how long it takes to correctly fit the matching substrate, then attempt to fit two non-matching substrates into the same rigid shape.
2. Induced fit model: Mould a piece of clay into an approximate active site shape. Press the same matching substrate into the clay and observe how the clay reshapes around it to form a snug fit. Repeat with the non-matching substrates, noting whether the clay can also accommodate a reasonable range of shapes.
3. For each model, record whether the correct substrate bound successfully, whether either incorrect substrate could also bind, and how long fitting took.
4. Remove each substrate and check whether the active site, card or clay, returns to a shape that can accept the correct substrate again, modelling enzyme reusability.
5. Compare both models against the interactive enzyme-substrate diagram earlier in this lesson and the X-ray crystallography evidence described for the induced fit model.

Discussion Questions

1. Which model, the rigid card or the reshaping clay, more accurately represents real enzyme behaviour? Justify your answer using the X-ray crystallography evidence described earlier in this lesson.
2. Explain one limitation of using a physical model to represent a molecular-level process such as enzyme-substrate binding.
3. Despite being an oversimplification, explain why the lock-and-key model is still a useful starting point for explaining enzyme specificity.
4. Suggest one modification to the clay model that would better represent the reversible nature of enzyme-substrate binding (E + S ⇌ ES).