Biology (9700) | Factors that affect enzyme action

Factors That Affect Enzyme Action: Comprehensive Study Notes (9700)

Welcome, Biologists! This chapter is crucial because enzymes control virtually every reaction in a living organism. Understanding how their speed (rate) is controlled—by environmental conditions and chemical factors—is key to grasping metabolism, genetic diseases, and biotechnology.
Don't worry if concepts like V_max or K_m seem intimidating; we will break them down using clear analogies!

1. Recapping Enzyme Function and Rate

Remember, enzymes are biological catalysts, usually globular proteins, that speed up reactions by lowering the Activation Energy. The rate of reaction is the speed at which substrates are converted into products.

How do we measure the rate of enzyme reactions?

In experiments (like those involving catalase or amylase), we typically measure the rate by tracking two things:

• Formation of Product: Measuring the volume of product released over time (e.g., oxygen produced by the enzyme catalase).
• Disappearance of Substrate: Measuring how quickly the substrate is used up (e.g., timing the disappearance of starch using the iodine test or measuring colour change using a Colorimeter).

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2. Physical Factors (Temperature and pH)

These two physical factors affect the enzyme's structure, which dictates its function.

2.1. The Effect of Temperature

Temperature affects both the movement of molecules and the structure of the enzyme itself.

A. Low Temperatures:
At low temperatures, the reaction rate is slow because the enzyme and substrate molecules have very little kinetic energy. This means they move slowly, resulting in a low frequency of successful collisions between the substrate and the active site.

B. Increasing Temperatures (Up to Optimum):
As temperature increases, kinetic energy increases. This leads to:

• More rapid movement of enzyme and substrate molecules.
• Increased frequency of effective collisions.
• Faster formation of the enzyme-substrate complex.
• The rate of reaction increases exponentially.

C. Optimum Temperature:
This is the temperature at which the enzyme is most active and the rate of reaction is at its maximum.

D. High Temperatures (Above Optimum):
If the temperature goes too high, the kinetic energy becomes so great that it starts to break the weak bonds (like hydrogen bonds, ionic bonds, and hydrophobic interactions) holding the enzyme's tertiary structure together.

• The enzyme loses its precise 3D shape.
• Crucially, the shape of the active site is destroyed.
• The substrate can no longer fit into the active site, destroying the enzyme's specificity.
• This permanent change is called Denaturation. The reaction rate falls rapidly to zero.

Analogy: Imagine a delicate ceramic key (the enzyme) melting in intense heat. It can no longer fit the lock (the substrate).

2.2. The Effect of pH

pH measures the concentration of hydrogen ions (\(H^{+}\)). Changes in pH primarily affect the charges on the R-groups (side chains) of the amino acids within the enzyme's structure, especially those involved in forming the active site.

A. Optimum pH:
Each enzyme has an optimum pH where its active site maintains the ideal ionic charges to attract and bind the substrate. For most intracellular enzymes, this is around neutral pH (pH 7).

Did you know? Enzymes operating outside cells often have different optima. For instance, the protease enzyme Pepsin, found in the stomach, works best around pH 2 (highly acidic)!

B. Deviating from Optimum pH:

• Adding excess \(H^{+}\) (low pH) or \(OH^{-}\) (high pH) disrupts the ionic bonds and hydrogen bonds holding the tertiary structure.
• The structure changes, leading to the distortion of the active site.
• If the pH change is small, the effect might be reversible if the original pH is restored. However, extreme pH changes cause permanent denaturation.

Syllabus Requirement: Using Buffer Solutions
When investigating the effect of pH, it is essential to use buffer solutions. A buffer is a mixture that resists changes in pH when small amounts of acid or alkali are added, ensuring the pH remains constant throughout the experiment.

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3. Concentration Factors

These factors relate to the availability of the molecules needed for the reaction.

3.1. Enzyme Concentration ([E])

If the substrate concentration is not limiting (i.e., there is plenty of substrate available), the rate of reaction is directly proportional to the enzyme concentration.

• Reason: Increasing the number of enzyme molecules provides more active sites. This increases the chance of successful collisions and therefore increases the rate of forming enzyme-substrate complexes.

Analogy: If you hire more toll booth operators (enzymes), more cars (substrates) can be processed per minute.

3.2. Substrate Concentration ([S])

Substrate concentration has a crucial effect on the reaction rate, shown by a characteristic curve on a graph (rate vs. [S]).

1. Initial Phase (Rate increases rapidly): When substrate concentration is low, the rate is proportional to [S]. There are many vacant active sites, and increasing the number of substrates increases the collision frequency.
2. Plateau Phase (Rate levels off): As [S] continues to increase, the curve flattens out. The reaction rate has reached its maximum, known as \(V_{max}\).

Why does the rate plateau?
The enzyme molecules become saturated with substrate. Every active site is constantly occupied. The enzyme concentration itself becomes the limiting factor—the enzymes cannot work any faster, even if more substrate is added.

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4. Advanced Kinetics: \(V_{max}\) and \(K_m\) (A Level Content)

To quantify enzyme efficiency and affinity for a substrate, we use terms related to the maximum rate (\(V_{max}\)).

4.1. Maximum Rate (\(V_{max}\))

\(V_{max}\) is the maximum rate of reaction that occurs when the substrate concentration is high enough to completely saturate all enzyme active sites. At this point, the enzyme concentration is the limiting factor.

4.2. Michaelis–Menten Constant (\(K_m\))

The Michaelis–Menten constant (\(K_m\)) is defined as the substrate concentration required to achieve half of the maximum reaction rate (\(0.5 \times V_{max}\)).

The value of \(K_m\) tells us about the enzyme’s affinity for its substrate:

• Low \(K_m\) Value: The enzyme only needs a low concentration of substrate to reach half its maximum speed. This indicates a high affinity for the substrate (they bind tightly).
• High \(K_m\) Value: The enzyme needs a high concentration of substrate to reach half its maximum speed. This indicates a low affinity for the substrate (they don't bind as readily).

Memory Aid: If \(K_m\) is Keep Me Low, the Affinity is High!

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5. Enzyme Inhibition

Inhibitors are molecules that reduce the rate of enzyme-catalysed reactions. We focus on reversible inhibitors which can dissociate from the enzyme.

5.1. Competitive Inhibition

Mechanism:
A competitive inhibitor has a molecular shape very similar to the normal substrate.
It competes with the substrate for the active site. If the inhibitor binds, it blocks the substrate from entering.

Analogy: This is like a substitute key that looks similar to the real key and gets stuck in the lock (active site), preventing the correct key (substrate) from entering.

Effects on Kinetics:

• \(V_{max}\): Unchanged. If you increase the substrate concentration dramatically, the substrate molecules will eventually outcompete the inhibitor, meaning the maximum potential rate is still reachable.
• \(K_m\): Increased. More substrate is required to reach half \(V_{max}\), indicating that the apparent affinity of the enzyme for the substrate has decreased.

5.2. Non-Competitive Inhibition

Mechanism:
A non-competitive inhibitor binds to a site on the enzyme that is not the active site. This site is called the allosteric site.
Binding to the allosteric site causes a change in the enzyme's tertiary structure, which distorts the shape of the active site, making it non-functional or less effective, even if the substrate is bound.

Analogy: This is like applying glue to the internal mechanism of the lock (allosteric site). The keyhole (active site) is still there, but the lock is broken and cannot function, regardless of how many keys you try.

Effects on Kinetics:

• \(V_{max}\): Decreased. The inhibitor reduces the number of functional enzyme molecules available. You cannot fix this by adding more substrate.
• \(K_m\): Unchanged. The inhibitor only removes functional enzyme molecules. The affinity of the remaining functional enzymes for the substrate is not altered.

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6. Immobilised Enzymes

In industrial and laboratory processes, enzymes are often fixed or trapped within an inert matrix, rather than being free in a solution. This process is called Immobilisation.

6.1. The Process (Investigation Context)

A common method of immobilisation involves trapping the enzyme (like yeast or catalase) within small, inert spheres, often made of calcium alginate. The substrate solution is then poured over these beads.

Investigation: By comparing the reaction rate of the enzyme immobilised in alginate versus the same enzyme free in solution, we can observe differences in activity and stability.

6.2. Key Advantages of Using Immobilised Enzymes

Immobilisation offers significant practical benefits for industry:

1. Reusability: The enzyme can be recovered easily at the end of the reaction and used repeatedly, saving money and reducing waste.
2. Easy Separation: The enzyme beads are physically separated from the product solution (e.g., via simple filtration or pouring), ensuring the product is pure.
3. Increased Stability: Being fixed within a matrix offers protection against environmental changes. Immobilised enzymes often show greater resistance to denaturation by changes in temperature and pH.
4. Continuous Process: Immobilised enzymes allow for a continuous flow system, where substrate is passed through a column of beads and product is continuously collected.

Key Takeaway: Immobilisation makes enzyme reactions far more cost-effective and controllable in industrial settings.