Metabolism is the sum of all the chemical reactions occurring within a living organism. These reactions must occur rapidly and under highly controlled conditions to sustain life. Left to themselves, most biological molecules are highly stable; they would react far too slowly at body temperature to keep an organism alive. This is where enzymes play a vital role.

What you'll learn

• How enzymes function as biological catalysts to control metabolism at both cellular and whole-organism levels.
• The mechanism of enzyme action, including the transition from the lock-and-key model to the induced-fit model.
• How physical and chemical factors affect the rate of enzyme-controlled reactions, and how to calculate the temperature coefficient ($Q_{10}$).
• The essential roles of cofactors, coenzymes, and different types of inhibitors in regulating enzyme activity.

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1. The Role of Enzymes in Metabolism

Enzymes are globular proteins that act as biological catalysts. They increase the rate of chemical reactions without being permanently altered or consumed in the process.

Structure and Function

Enzymes do not just determine functional processes; they also determine cellular and physical structures.

• Structural roles: Enzymes catalytically synthesise structural polymers. For example, the enzyme collagen lysyl oxidase cross-links collagen fibres, giving bone, cartilage, and blood vessels their structural strength and elasticity.
• Functional roles: Enzymes catalyse the metabolic pathways that drive active transport, muscle contraction, and cell signalling.

Intracellular vs. Extracellular Enzymes

Not all enzymes operate in the same environment. Some must stay inside the cytoplasm or organelles, while others are secreted to work outside cells.

• Intracellular enzymes catalyse reactions inside the cell. A key example is catalase. Respiration and other metabolic pathways produce toxic hydrogen peroxide ($H_2{O}_2$) as a byproduct. Catalase rapidly converts this toxic chemical into harmless water ($H_{2}O$) and oxygen ($O_2$), protecting the cell from oxidative damage.
• Extracellular enzymes are synthesised inside the cell but are secreted to catalyse reactions outside the cell. For example, amylase is secreted by the salivary glands and pancreas into the mouth cavity and duodenum to catalyse the hydrolysis of insoluble starch into soluble maltose. Another example is trypsin, a protease secreted by the pancreas to digest proteins in the small intestine.

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2. The Mechanism of Enzyme Action

To understand how enzymes speed up reactions, we must look at their tertiary structure. Because enzymes are proteins, their specific three-dimensional shape is dictated by the primary sequence of amino acids, which determines the folding and bonding (hydrogen bonds, ionic bonds, disulfide bridges, and hydrophobic interactions).

Lowering Activation Energy

Chemical reactions require a certain amount of energy to start. This is the energy needed to break bonds in the reactants so that new bonds can form.

Enzymes speed up chemical reactions by lowering the activation energy. They do this by holding the substrate molecules close together or putting physical strain on their chemical bonds, making them much easier to break.

Hypotheses of Enzyme Action

Historically, scientists explained enzyme specificity using the lock-and-key hypothesis. Over time, further experimental evidence led to the development of the induced-fit hypothesis.

The Lock-and-Key Hypothesis

This model suggests that the enzyme's active site is a rigid, fixed shape (the "lock"). The substrate has a complementary shape (the "key") that fits perfectly into the active site.

1. The substrate collides successfully with the active site.
2. They bind to form an enzyme-substrate complex (ESC).
3. The chemical reaction occurs, temporarily forming an enzyme-product complex (EPC).
4. The products leave the active site, leaving the enzyme unchanged and ready to accept another substrate.

The Induced-Fit Hypothesis

While the lock-and-key model explains specificity, it does not explain how the binding process actually lowers the activation energy. The induced-fit hypothesis addresses this. It suggests that the active site is not completely rigid, but flexible.

1. The substrate collides with the active site.
2. As the substrate binds, it induces a conformational change (a slight change in shape) in the active site of the enzyme.
3. The active site moulds itself tightly around the substrate.
4. This conformational change puts physical strain on the bonds of the substrate, destabilising them and lowering the activation energy.
5. Once the reaction is complete, the products have a different shape, no longer fit the active site, and diffuse away.

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3. Factors Affecting Enzyme Activity

The rate of an enzyme-controlled reaction depends heavily on the physical and chemical conditions of its environment.

Temperature

As temperature increases, the kinetic energy of the enzyme and substrate molecules increases. They move faster, resulting in more frequent, successful collisions. This leads to a higher rate of enzyme-substrate complex formation.

However, if the temperature rises above an optimum point, the thermal energy makes the atoms within the enzyme vibrate so rapidly that the weak hydrogen and ionic bonds stabilizing the tertiary structure break. The active site changes shape, losing its complementary relationship with the substrate. The enzyme is said to be denatured.

The Temperature Coefficient ($Q_{10}$)

We can quantify the effect of temperature on the rate of reaction using the temperature coefficient, $Q_{10}$.

The formula for calculating $Q_{10}$ is:

$$Q_{10}=\frac{R_2}{R_1}$$

where:

• $R_2$ is the rate of reaction at temperature $(T+10{)}^{\circ }C$
• $R_1$ is the rate of reaction at temperature $T^{\circ }C$

For most enzyme-controlled reactions below their optimum temperature, the rate of reaction approximately doubles for every $10^{\circ }C$ rise in temperature, giving a $Q_{10}$ value of approximately 2.

pH

pH is a measure of the hydrogen ion ($H^{+}$) concentration. $H^{+}$ ions have a positive charge. If the pH deviates from the optimum value for a specific enzyme, the excess $H^{+}$ ions (in acidic conditions) or lack of them (in alkaline conditions) will interact with the charged R-groups of the amino acids in the active site.

This disrupts the hydrogen and ionic bonds holding the tertiary structure together, altering the shape of the active site and preventing the substrate from binding. Extreme changes in pH lead to denaturation.

Enzyme and Substrate Concentration

• Substrate concentration: As substrate concentration increases, the rate of reaction increases because there are more substrate molecules to collide with the active sites. However, at a certain point, the rate plateaus at a maximum velocity ($V_{max}$). At this point, all the active sites are saturated with substrate. Enzyme concentration has become the limiting factor.
• Enzyme concentration: Similarly, as enzyme concentration increases, the rate increases because there are more active sites available. Eventually, the rate plateaus when there are not enough substrate molecules to bind to the excess active sites. Here, substrate concentration becomes the limiting factor.

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4. Practical Skills: Serial Dilutions

In practical investigations of enzyme activity (such as PAG 4), you are often required to make a range of concentrations of a substrate or an enzyme from a concentrated stock solution. A highly precise way to do this is through a serial dilution.

To perform a 2-fold (halving) serial dilution:

1. Set up a row of five clean test tubes.
2. Add $10{cm}^3$ of distilled water to tubes 2, 3, 4, and 5.
3. Add $20{cm}^3$ of your stock solution (e.g., $2.0\%$ yeast suspension) to tube 1.
4. Transfer $10{cm}^3$ of yeast suspension from tube 1 into tube 2 using a clean syringe. Mix thoroughly. Tube 2 now has a concentration of $1.0\%$.
5. Repeat this step-by-step transfer of $10{cm}^3$ from tube 2 to 3 ($0.5\%$), then tube 3 to 4 ($0.25\%$), and tube 4 to 5 ($0.125\%$). Finally, discard $10{cm}^3$ from tube 5 so all tubes contain equal volumes.

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5. Cofactors and Coenzymes

Many enzymes require the presence of non-protein helper molecules to carry out their catalytic function.

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6. Enzyme Inhibition

Enzymes can be switched off or slowed down by molecules called inhibitors. This is crucial for controlling metabolic pathways and is also the basis of many poisons and medicines.

Competitive vs. Non-Competitive Inhibitors

Competitive Inhibitors

These molecules have a similar molecular shape to the substrate.

• They bind temporarily to the active site, physically blocking the substrate from entering.
• They do not damage the active site permanently.
• Overcoming inhibition: If you drastically increase the concentration of the substrate, the substrate molecules outcompete the inhibitor molecules. The maximum rate of reaction ($V_{max}$) can still be reached, but it requires a much higher substrate concentration.

Non-Competitive Inhibitors

These molecules do not bind to the active site. Instead, they bind to an alternative site on the enzyme known as the allosteric site.

• Binding to the allosteric site alters the tertiary structure of the enzyme, changing the shape of the active site.
• The active site is no longer complementary to the substrate, so the substrate can no longer bind.
• Overcoming inhibition: Adding more substrate has no effect because the active sites are permanently altered. The reaction will never reach the original $V_{max}$.

Reversible vs. Irreversible Inhibitors

• Reversible inhibitors bind weakly (e.g., via hydrogen or ionic bonds) and can dissociate from the enzyme.
• Irreversible inhibitors form strong covalent bonds with the enzyme, permanently disabling it. Many highly toxic poisons (like cyanide, which inhibits cytochrome c oxidase in mitochondria) work as irreversible inhibitors.

End-Product Inhibition

Cells use end-product inhibition to control metabolic pathways through a negative feedback loop.

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