Biology (9700) | Enzymes

Welcome to the Wonderful World of Enzymes!

Hello future Biologists! This chapter is absolutely fundamental. Enzymes are the tiny biological machines that make life possible—they control almost every chemical reaction in your body, from digesting your lunch to making new DNA.
If you find biochemistry challenging, don't worry! We will break down these concepts using simple analogies and clear steps. Master this topic, and you’ll have a huge advantage when studying respiration and photosynthesis later!

3.1 Mode of Action of Enzymes

What Exactly is an Enzyme? (A Quick Definition)

An enzyme is a biological catalyst.

• Catalyst: A substance that increases the rate of a chemical reaction without being used up or changed itself.
  
• Biological: Enzymes are made inside living organisms. They are globular proteins.

Intracellular vs. Extracellular Enzymes

Enzymes work both inside and outside cells:

• Intracellular Enzymes: Work inside the cell where they were made (e.g., catalase, which breaks down toxic hydrogen peroxide, or the enzymes used in respiration).
• Extracellular Enzymes: Are secreted out of the cell to catalyse reactions outside (e.g., digestive enzymes like amylase, which breaks down starch in the mouth and gut).

The Mechanism of Enzyme Action: The Key Players

All enzyme action revolves around three core components:

1. Active Site

The active site is a specific region on the surface of the enzyme molecule.

• It has a specific 3D shape, determined by the enzyme's tertiary structure (the way the polypeptide chain is folded).
• This site is where the reactant molecule, called the substrate, binds.

2. Substrate

The substrate is the molecule on which the enzyme acts.

• Enzymes show enzyme specificity: usually, only one type of substrate can fit and react with a particular enzyme. This is essential for controlling metabolic pathways.

3. Enzyme-Substrate Complex (ESC)

When the substrate molecule binds temporarily to the active site, it forms an enzyme-substrate complex (ESC).

• The reaction occurs while the substrate is held in the active site.
• Once the reaction is complete, the products are released, and the enzyme is free to bind with a new substrate molecule.

Lowering the Activation Energy

The main job of an enzyme is to increase the rate of reaction by lowering the activation energy (AE).

• Activation Energy (AE): The minimum amount of energy required for a chemical reaction to start.
• Enzymes provide an alternative reaction pathway that requires less energy.

Analogy: Imagine you need to push a heavy boulder over a hill (the AE barrier) to get it to the other side. An enzyme acts like a tunnel carved straight through the hill, requiring much less effort (energy) to move the boulder (substrate).

Models of Enzyme Specificity

a) The Lock-and-Key Hypothesis

This was the first model proposed (by Emil Fischer in 1894).

• It suggests the active site has a rigid shape, perfectly complementary to the substrate, like a key fitting into a specific lock.
• Limitation: This model suggests the enzyme is completely static, which isn't quite accurate based on modern evidence.

b) The Induced-Fit Hypothesis

This is the modern and more accepted theory (proposed by Daniel Koshland in 1958).

• The active site is not rigid; it is flexible.
• When the substrate enters the active site, it causes a slight conformational change (a change in shape) in the enzyme.
• This subtle shape change allows the active site to mould itself more tightly around the substrate, ensuring a perfect fit and putting strain on the substrate bonds, which helps lower the AE.

Memory Aid: Think of the substrate inducing (causing) the enzyme to change shape, like a hand fitting perfectly into a soft glove—it's snug, flexible, and gets the job done better!

3.2 Factors that Affect Enzyme Action

The rate at which an enzyme works is highly dependent on its environment because these factors can change the enzyme's 3D shape (its tertiary structure).

1. Temperature

Temperature changes affect the kinetic energy of both enzyme and substrate molecules.

• Low Temperatures: Low kinetic energy means fewer effective collisions between the enzyme and substrate, resulting in a very slow reaction rate.
• Optimum Temperature: This is the temperature at which the enzyme is working at its maximum rate (\(V_{max}\)). For most human enzymes, this is around 37 °C.
• Increasing Temperature (above optimum):
  1. Increased kinetic energy causes the enzyme molecules to vibrate violently.
  2. This vibration breaks the weak bonds (hydrogen bonds, ionic bonds) that maintain the specific 3D structure of the active site.
  3. The active site changes shape, meaning the substrate can no longer bind.
  4. The enzyme has permanently lost its functional shape—this is called denaturation. Denaturation is irreversible, causing the reaction rate to drop sharply to zero.

Common Mistake to Avoid: Heating an enzyme above optimum causes denaturation, not just inactivation. Cooling an enzyme down inactivates it, but the shape remains intact, and activity can recover upon warming.

2. pH (Hydrogen Ion Concentration)

pH affects the charged groups (R-groups) of the amino acids that make up the active site, particularly the ionic bonds.

• Optimum pH: Each enzyme has a specific pH where it is most active (e.g., pepsin in the stomach works best around pH 2; catalase works best around pH 7).
• Deviation from Optimum: If the pH is too high (alkaline) or too low (acidic), the excess H+ or OH- ions interfere with the ionic bonds and hydrogen bonds holding the tertiary structure together.
• This causes the active site to change shape, reducing or eliminating substrate binding (denaturation).

Practical Note: In investigations involving pH, buffer solutions must be used. A buffer solution resists changes in pH, ensuring the reaction mixture maintains a constant H+ concentration throughout the experiment.

3. Enzyme Concentration

Assuming the substrate supply is plentiful:

• If you double the enzyme concentration, you double the rate of reaction.
• This is because there are more active sites available for substrates to bind to, increasing the frequency of successful collisions and the formation of ESCs.
• The rate is directly proportional to the enzyme concentration.

4. Substrate Concentration

Assuming the enzyme concentration is constant:

• Low Concentration: An increase in substrate concentration leads to a rapid increase in the reaction rate, as more active sites are occupied.
• High Concentration (Saturation): Eventually, adding more substrate will have no effect on the rate. This is because all active sites are saturated (full). The enzymes are working as fast as they can (at their maximum rate, \(V_{max}\)). The enzyme activity is now the limiting factor.

Advanced Concepts in Enzyme Kinetics (A-Level Focus)

Maximum Rate (\(V_{max}\)) and Affinity (\(K_m\))

In enzyme studies, we use specific terms to describe enzyme efficiency:

• Maximum Rate (\(V_{max}\)): The fastest rate at which an enzyme can catalyse a reaction when the substrate concentration is so high that all active sites are saturated.
• Michaelis-Menten Constant (\(K_m\)): This constant is the substrate concentration required to achieve half of the maximum velocity (\(V_{max} / 2\)).

What \(K_m\) tells us:

• A low \(K_m\) indicates that the enzyme only needs a small amount of substrate to become half-saturated, meaning it has a high affinity for the substrate. (A good, tight bond).
• A high \(K_m\) indicates the enzyme needs a lot of substrate to reach half its maximum rate, meaning it has a low affinity for the substrate. (A weak, slow bond).

Inhibitors: Slowing Down the Action

What are Inhibitors?

Inhibitors are molecules that slow down or stop an enzyme-catalysed reaction. They are crucial for metabolic control and are the basis of many medical drugs.

We focus on reversible inhibitors, which do not permanently damage the enzyme.

1. Competitive Inhibition

These inhibitors compete with the substrate for the active site.

• Structure: They have a shape very similar to the enzyme’s natural substrate.
• Effect: They temporarily block the active site, preventing the formation of the ESC.
• Overcoming the effect: This inhibition can be overcome by increasing the substrate concentration. If the substrate concentration is much higher than the inhibitor concentration, the substrate molecules are more likely to collide with and occupy the active site.
• Kinetics: Competitive inhibitors increase the \(K_m\) (lower affinity) but do not change the \(V_{max}\) (the enzyme can still reach full speed if enough substrate is present).

Analogy: Competitive inhibitors are like siblings trying to sit in the same chair as you. If you bring more siblings, they still have to compete for the single chair (active site). If you flood the area with your substrate (you!), you win.

2. Non-Competitive Inhibition

These inhibitors bind to a site other than the active site.

• Binding Site: They bind to the allosteric site (a different location on the enzyme molecule).
• Effect: Binding at the allosteric site changes the overall tertiary structure of the enzyme, including the shape of the active site. This means the active site can no longer bind the substrate effectively, regardless of how much substrate is present.
• Overcoming the effect: This inhibition cannot be overcome by increasing substrate concentration, because the active site is physically deformed.
• Kinetics: Non-competitive inhibitors decrease the \(V_{max}\) (because fewer functional enzyme molecules are available) but do not change the \(K_m\) (the remaining functional enzymes still have the same affinity).

Analogy: Non-competitive inhibitors are like a spanner thrown into the machine (the enzyme). It doesn't block the door (active site) but twists the whole machine, rendering the door unusable.

3.2 (continued) Applications of Enzymes

Immobilised Enzymes

Enzymes used in industrial processes (like making lactose-free milk or high-fructose corn syrup) are often physically attached to an inert material. This is called enzyme immobilisation.

A common method for investigation involves trapping the enzyme (e.g., lactase) within tiny beads of an inert gel, such as alginate (derived from seaweed).

Advantages of using Immobilised Enzymes

Using enzymes fixed to a surface offers several significant advantages over using enzymes free in solution:

1. Reusability: The immobilised enzymes can be used repeatedly in a continuous flow system, significantly reducing production costs.
2. Easy Separation: The enzymes are easily separated from the product simply by filtering the alginate beads. This prevents contamination and simplifies downstream purification.
3. Increased Stability: Being trapped in the matrix makes the enzymes more stable and less prone to denaturation due to heat or pH changes.
4. Controllable Reactions: The reaction can be instantly stopped by simply halting the flow of the substrate over the enzyme column.

Example: Immobilised lactase is used to break down lactose into glucose and galactose, making milk digestible for lactose-intolerant people.

Key Takeaway: Immobilisation

Immobilising enzymes (e.g., in alginate) makes them much better for industry because they are stable, easy to separate, and can be reused many times, saving money!