Welcome to AS 1 Biology: Enzymes!
Welcome to one of the most exciting and fundamental topics in AS Level Biology! Whether you love biochemistry or find molecules a bit daunting, don't worry. We will break down every single concept step-by-step.
In this chapter, you will learn how biological catalysts work, what speeds them up or slows them down, how inhibitors interfere with them, and how scientists trap enzymes in industrial beads to produce products efficiently. Let's dive in!
1. Nature and Mode of Action of Enzymes
What is an Enzyme?
An enzyme is a biological catalyst that speeds up the rate of a metabolic chemical reaction without being used up or permanently changed in the process.
Metabolism: Building Up and Breaking Down
All chemical reactions happening inside a living organism make up its metabolism. Metabolic reactions fall into two categories:
• Anabolic reactions: Building larger, complex molecules from smaller units. These require an input of energy (endergonic). Think: Anabolic steroids build muscle mass.
• Catabolic reactions: Breaking down complex molecules into simpler products. These release energy (exergonic). Think: A catastrophe breaks things apart.
Protein Structure of Enzymes
Enzymes are globular proteins. They have a specific tertiary structure (and sometimes a quaternary structure) that folds the polypeptide chain into a precise three-dimensional shape. Within this structure is a small, specialized cleft or pocket called the active site.
The active site is where the reactant molecule—known as the substrate—binds during a reaction.
How Enzymes Work: Lowering Activation Energy
Chemical reactions need a kickstart to begin. The minimum amount of energy required to start a reaction is called the activation energy (\(E_a\)).
Enzymes speed up reactions by lowering the activation energy. They provide an alternative reaction pathway, allowing bonds within the substrate to break or form much more readily at normal body temperatures.
Models of Enzyme Action
Scientists use two models to explain how an enzyme interacts with its substrate:
1. The Lock-and-Key Model (Fischer):
This model suggests that the shape of the enzyme's active site is rigid and an exact complementary fit to the substrate, just like a key fitting precisely into a lock.
2. The Induced-Fit Model (Koshland):
This model gives a more accurate description. The active site is flexible. As the substrate approaches and enters the active site, it induces a conformational (shape) change in the enzyme. The active site moulds closely around the substrate, putting strain on the substrate's chemical bonds and facilitating catalysis. Once the products leave, the active site reverts to its original shape.
During the reaction, the sequence of events is:
Enzyme + Substrate \(\rightarrow\) Enzyme-Substrate Complex (ESC) \(\rightarrow\) Enzyme-Product Complex (EP) \(\rightarrow\) Enzyme + Product
Common Examiner Pitfall: Denaturation
Warning: Never say an enzyme is "killed" or "dead"! Enzymes are non-living chemical molecules. Under extreme heat or pH, say: "The hydrogen and ionic bonds maintaining the tertiary structure break, altering the 3D shape of the active site so it is no longer complementary to the substrate."
Key Takeaway
Enzymes are globular proteins with a specific 3D active site. They lower activation energy (\(E_a\)) by forming enzyme-substrate complexes (ESCs) via an induced-fit mechanism.
2. Co-factors, Prosthetic Groups, and Coenzymes
Many enzymes require helper substances to function properly. These non-protein components are grouped as follows:
• Co-factors: A general umbrella term for any non-protein chemical substance needed for an enzyme to carry out its catalytic activity.
• Prosthetic Groups: Non-protein helper molecules that are permanently and covalently attached to the enzyme's polypeptide chain (for example, the iron-containing haem group in certain enzymes).
• Coenzymes: Small, non-protein organic molecules (often derived from vitamins) that are transiently (temporarily) bound to the enzyme, participating directly in the reaction cycle before dissociating.
Key Takeaway
Prosthetic groups are permanently attached structural helpers; coenzymes are temporarily associated organic helpers.
3. Factors Affecting the Rate of Enzyme Reactions
A. Temperature
• Rising temperature up to optimum: As temperature rises, thermal energy is converted to kinetic energy. Enzymes and substrates move faster, colliding more frequently and with greater force. This leads to more successful collisions per second and a higher rate of enzyme-substrate complex (ESC) formation.
• Optimum temperature: The temperature at which the rate of reaction is at its maximum.
• Above optimum temperature: Increased thermal vibrations break the delicate hydrogen and ionic bonds holding the enzyme's tertiary structure in place. The active site loses its specific 3D shape—the enzyme is denatured. The substrate can no longer bind to form an ESC, and the rate drops rapidly to zero.
B. pH
• Every enzyme has an optimum pH where its active site has the ideal complementary shape.
• If the pH deviates (becomes too acidic or too alkaline), excess \(H^+\) or \(OH^-\) ions interact with the charged R-groups of the amino acids forming the active site.
• This disrupts the ionic and hydrogen bonds, changing the tertiary structure and denaturing the enzyme.
C. Substrate Concentration
• At low substrate concentrations, substrate molecules are the limiting factor. Many active sites remain unoccupied.
• Adding more substrate increases successful collisions and ESC formation, raising the rate.
• Eventually, a plateau is reached called the maximum velocity (\(V_{\max}\) or saturation point). At this stage, all active sites are continuously occupied. The enzyme concentration is now the limiting factor.
D. Enzyme Concentration
• As enzyme concentration increases, there are more active sites available to form ESCs.
• Provided that substrate is in excess (not limiting), the rate of reaction is directly proportional to enzyme concentration.
Calculating the Rate of Reaction
You may be asked to calculate the rate from experimental data in two ways:
1. Simple relative rate: \(\text{Rate} = \frac{1}{\text{Time}}\)
2. Initial Rate from a Graph: When plotting product formed (or substrate used) against time, measure the initial linear portion. Draw a tangent to the curve at time = 0 and calculate the gradient:
\(\text{Gradient} = \frac{\Delta y}{\Delta x}\)
Exam Tip: Always use the initial rate! Later in the reaction, the rate slows down because the substrate is being used up (substrate depletion).
Key Takeaway
Reaction rates depend on kinetic energy (temperature), active site charge/shape (pH), and collision frequency (concentrations). Rates level off at \(V_{\max}\) when all active sites are saturated.
4. Enzyme Inhibition
An inhibitor is any substance that slows down or stops an enzyme-catalysed reaction.
A. Competitive Inhibition
• Structure: The inhibitor has a molecular shape that is similar or complementary to the substrate.
• Binding Site: It binds directly to the active site of the enzyme, blocking the substrate from entering.
• Overcoming the effect: Because it competes with the substrate, you can overcome competitive inhibition by increasing substrate concentration. A higher substrate-to-inhibitor ratio means substrate molecules outcompete the inhibitor for active sites.
• Effect on \(V_{\max}\): \(V_{\max}\) can still be reached, but requires a higher substrate concentration.
B. Non-Competitive Inhibition
• Structure: The inhibitor does not need to resemble the substrate.
• Binding Site: It binds to an allosteric site (a site away from the active site).
• Mechanism: Binding distorts the tertiary structure of the enzyme, changing the 3D shape of the active site so the substrate no longer fits.
• Overcoming the effect: It cannot be overcome by adding more substrate because the affected enzymes are rendered inactive.
• Effect on \(V_{\max}\): \(V_{\max}\) is permanently lowered.
Reversible vs. Irreversible Inhibition
• Reversible inhibition: Inhibitors bind via weak bonds (e.g. hydrogen bonds) and can detach.
• Irreversible inhibition: Inhibitors form strong, permanent covalent bonds with the enzyme, permanently inactivating it.
Key Takeaway
Competitive inhibitors bind to the active site (overcome by adding substrate; \(V_{\max}\) unchanged). Non-competitive inhibitors bind to an allosteric site (cannot be overcome; \(V_{\max}\) reduced).
5. Immobilised Enzymes in Biotechnology
What are Immobilised Enzymes?
Immobilised enzymes are enzymes that are physically attached to or entrapped within an inert, insoluble support material rather than floating freely in solution.
The Four CCEA Methods of Immobilisation
1. Adsorption: Enzymes are attached by weak physical forces (electrostatic attractions or hydrogen bonds) to the surface of an inert solid carrier, such as glass, clay, or collagen.
2. Cross-linking / Covalent Bonding: Enzymes are chemically bound via strong covalent bonds to an insoluble matrix (like cellulose) or cross-linked directly to one another using chemical agents.
3. Entrapment: Enzymes are trapped within the microscopic pores of an insoluble gel lattice (such as calcium alginate beads). Substrates and products diffuse freely in and out, but the larger enzyme molecules cannot escape.
4. Encapsulation (Membrane Confinement): Enzymes are enclosed behind a semi-permeable membrane or inside microscopic nylon capsules.
Advantages of Using Immobilised Enzymes
• Increased Thermostability & pH Resistance: The supporting matrix holds the enzyme's tertiary structure rigidly, making it less susceptible to denaturation at higher temperatures and extreme pH levels.
• Reusability: The enzymes can be easily recovered and reused multiple times, drastically reducing production costs.
• Continuous Flow Processing: Substrate can be poured continuously over a packed column of immobilised enzymes, yielding continuous product output without stopping for batch cleanups.
• Product Purity: The product is not contaminated with enzyme molecules, eliminating expensive downstream purification and separation stages.
Disadvantages of Immobilised Enzymes
• Active sites may be partially blocked, shielded, or distorted during the immobilisation process.
• Diffusion limitations: Substrates take time to diffuse through the gel matrix or capsule membrane, which can make the reaction rate slower than with free enzymes in solution.
Key Takeaway
Immobilising enzymes (by adsorption, cross-linking, entrapment, or encapsulation) makes them more stable, reusable, and keeps the product pure, though diffusion through support matrices may slightly slow the reaction.
Quick Summary Checklist
Before your AS 1 exam, make sure you can:
✔ Define an enzyme, active site, and activation energy (\(E_a\)).
✔ Contrast the lock-and-key model with the induced-fit model.
✔ Differentiate between co-factors, prosthetic groups, and coenzymes.
✔ Explain how temperature, pH, substrate, and enzyme concentration alter reaction rates.
✔ Accurately define denaturation in terms of bond breakage and tertiary structure.
✔ Calculate reaction rates using \(\text{Rate} = \frac{1}{\text{Time}}\) and graph gradients (\(\frac{\Delta y}{\Delta x}\)).
✔ Compare competitive (active site) and non-competitive (allosteric site) inhibition.
✔ Describe the 4 methods and evaluate the commercial advantages/disadvantages of immobilised enzymes.