Enzymes (2.1.4)

Enzymes act as biological catalysts, lowering the activation energy required for a reaction to occur by forming weak interactions with the substrate. These interactions can strain the bonds in the substrate, making them more likely to break or form.

Enzymes are not altered or used up during a reaction; they can be reused.

Enzymes catalyse catabolic reactions where large molecules are broken down into smaller molecules. These reactions usually release energy (and the end-products can be used in respiration to produce ATP).

For example, the enzyme amylase catalyses the hydrolysis (breakdown) of starch into maltose.
Without enzymes catalysing catabolic reactions, large molecules in our food would not be broken down or energy released at a rate fast enough to support life.

Extracellular enzymes are secreted by cells to function outside of the cell. They play crucial roles in digestion, where large nutrient molecules need to be broken down or modified outside the cell.

For example, amylase is an enzyme produced by cells in the salivary glands and pancreas. When released into saliva, it catalyses the hydrolysis of starch (a polysaccharide) into maltose (a disaccharide).

Enzymes are globular proteins with a specific tertiary (3-dimensional) structure. An area within this structure, called the enzyme’s active site, is where substrates bind, forming an enzyme–substrate complex.

The shape of an enzyme’s active site is complementary to the substrate(s) involved in the specific reaction it catalyses.

The lock-and-key hypothesis of enzyme action suggests that a substrate fits into the rigid structure of an enzyme’s active site precisely, in the same way a specific key fits into its corresponding lock.

When an enzyme and its substrate collide, the side chains of amino acids (R groups) within the enzyme’s active site form temporary bonds with the substrate. This results in the formation of an enzyme–substrate complex.

The enzyme catalyses the reaction, and the products are formed in an enzyme–product complex. The enzyme then releases the products and is left unchanged, ready to be reused.

The induced fit model of enzyme action suggests that enzymes are flexible and can change shape in the presence of a substrate. These changes in shape are known as conformational changes.

As an enzyme and substrate collide, the side chains of amino acids (R groups) within the enzyme’s active site form temporary bonds with the substrate. These weak interactions induce a change in the enzyme’s tertiary structure, strengthening the binding in the enzyme–substrate complex. This strains the bonds in the substrate making them easier to break (lowering the activation energy required for the reaction).

The products are formed within an enzyme–product complex and are then released. The enzyme returns to its original shape, ready to be reused.

The rate of an enzyme-catalysed reaction can be measured from a graph of the amount of product formed or substrate used through time.

The gradient of the curve at any given point is equal to the rate of the reaction (units are always ‘per unit time’). This changes over time if the relationship is curved.

To investigate the effect of pH on enzyme activity, we can use the enzyme amylase to break down starch into maltose. The progress of the reaction is monitored using iodine as an indicator (iodine = yellow-brown; starch + iodine = blue-black).

Buffer solutions have a specific pH and maintain it, even if the reaction taking place would otherwise cause the pH of the reaction mixture to change.

Experimental procedure:

1. Place drops of iodine indicator solution (potassium iodide) into the wells of a spot plate.
2. Label a test tube with the buffer solution being used (pH levels to be tested).
3. Add of amylase solution to each test tube.
4. Add of the buffer solution to the amylase using a syringe.
5. Add of starch solution to the mixture, start the stopwatch, and mix.
6. Every 10 seconds, place a drop of the mixture onto a drop of iodine indicator solution on the spot plate.
7. Note the time when the iodine indicator no longer turns blue-black, suggesting that the starch has completely broken down. The faster the iodine indicator stops turning blue-black, the more effective the enzyme is at that pH.
8. Repeat for different buffer solutions (pH values).

To investigate the effect of temperature on enzyme activity, we can use the enzyme catalase which breaks down hydrogen peroxide into water and oxygen.

Experimental procedure:

1. Cut a set weight of raw potato.
2. Set up the equipment, as shown below.
3. Add the potato to the hydrogen peroxide, start the timer, and mix.
4. Record the volume of oxygen produced every 10 seconds.

Repeat this procedure with the same weight of potato that has been boiled for 10 minutes.

The temperature coefficient is a measure of the change in the rate of a reaction after increasing the temperature by .

is calculated using the formula:

where:
is the initial temperature.

For most enzyme-catalysed reactions, values typically range between 2 and 3. This means the rate of reaction roughly doubles or triples with every rise in temperature.

The relationship does not apply outside of an enzyme’s optimum temperature range.

Serial dilutions are useful for testing the impact of substrate or enzyme concentration on reaction rate. This dilution technique is used to produce a series of solutions with progressively lower concentrations.

Initially, increasing substrate concentration increases the rate of reaction. With more substrate molecules present, the chance of forming enzyme–substrate complexes increases.

The rate of reaction subsequently plateaus as all active sites become saturated. The rate of reaction reaches its maximum . Beyond this point, further increase in substrate concentration will not increase the reaction rate, as there are no free active sites for additional substrate molecules to bind to. The enzyme concentration becomes the limiting factor in the reaction.

Initially, increasing enzyme concentration increases the rate of reaction because there are more active sites available to form enzyme–substrate complexes. With ample substrate available, the initial reaction rate increases linearly with enzyme concentration.

If the substrate is limited, increasing the enzyme concentration further will not increase the reaction rate, because substrate availability becomes the limiting factor. The reaction rate will have reached

Coenzymes are small, organic molecules that help enzymes function by transferring chemical groups or electrons between reactions or by forming part of the active site. Although they are temporarily altered during reactions, they are continuously recycled.

Many coenzymes are derived from vitamins. For example:

• Vitamin B₅ (pantothenic acid) is a key component of coenzyme A.
• Vitamin B₃ (nicotinic acid) is used to produce the coenzymes NAD and NADP.
• Vitamin B₁ (riboflavin) is used to produce the coenzyme FAD.

Inhibitors are molecules which stop enzymes from functioning or slow them down. They can bind to enzymes reversibly or irreversibly. There are two types of inhibitors:

• Competitive inhibitors have a similar shape to the substrate and compete to bind to the enzyme’s active site. By forming enzyme–inhibitor complexes, competitive inhibitors reduce the formation of enzyme–substrate complexes.
• Non-competitive inhibitors bind to the enzyme at an alternative site (or allosteric site), which alters the shape of the active site. This reduces the activity of the enzyme or prevents the substrate from binding.

Competitive and non-competitive inhibitors decrease the reaction rate. At sufficiently high inhibitor concentrations, the reaction can cease entirely.

Increasing substrate concentration can counteract the effect of a competitive inhibitor, as more substrate molecules are available to collide with enzymes and form enzyme–substrate complexes.

Increasing substrate concentration does not restore the reaction rate in the presence of a non-competitive inhibitor, since the inhibitor changes the enzyme’s active site, preventing enzyme–substrate complex formation.

Metabolic pathways can be controlled through end-product inhibition, where the final product of a metabolic pathway acts as a non-competitive, reversible inhibitor.

As the end-product accumulates, it binds to an alternative site on the initial enzyme, altering its active site and reducing or stopping its catalytic activity by decreasing the formation of enzyme–substrate complexes.

If the end-product concentration falls, it detaches from the enzyme, restoring the enzyme’s activity. This feedback loop allows catalysis to resume as product levels decrease.