Enzyme reactions

• Reactions occur in sequences called metabolic pathways, which are controlled by enzymes. Products of one reaction are the reactants in another.

• Two examples of pathways are: anabolic reactions - the building up of molecules, e.g protein synthesis - and catabolic reactions - breaking down of molecules, e.g digestion.

• Metabolism being the sum of all reactions in the body: our bodies are at a basal metabolic rate ordinarily - this is the metabolism at rest, the lowest level of cellular function.

General properties of enzymes

• Enzymes are known as biological catalysts, as they are made by living cells.

• They are globular proteins - tertiary structure with hydrophilic R groups on the outside, making them soluble. Each enzyme has a different amino acid sequences.

• The active site is a small area with a specific 3D shape on an enzyme, and it gives enzymes many of its properties. This is the area where enzymes attach to a substrate.

• Enzymes speed up reactions, are not used up during reactions, do not change after reactions and have a high-turnover rate (catalyse many reactions per second).

• Enzymes only catalyse energetically favourable reactions that would happen without their involvement. However, without enzymes reactions would take too long, and it would be impossible for human life to function.

Sites of enzyme action

Enzymes are created inside cells, with three distinct sites where they act.

Extracellular:

These cells are secreted by exocytosis and catalyse extracellular reactions. An example is amylase, which is made in the salivary glands. Most extracellular reactions are digestive ones.

Intracellular, in solution

These enzymes act in cells within a solution. An example is glucose synthesis in the stroma.

Intracellular, membrane-bound

These enzymes are attached to membranes, such as the cristae in mitochondria where they transfer electrons and hydrogen atoms in ATP synthesis.

Enzyme models

Scientists have decided to show the action of enzymes through different models; the lock-and-key model and induced fit model. While lock-and-key is the oldest model, it is not entirely outdated and therefore still used by many scientists. This is due to the differing behaviour of enzymes - they can fit either model more closely.

Lock-and-key model

The enzyme is specifically shaped to always fit one type of substrate, so it can only catalyse one reaction. The term for this is enzyme specificity, that enzymes are specifically created to catalyse one reaction. To show this, the key is used to emulate a enzyme and the substrate is the lock; therefore the reaction is unlocked. An example is tyrosine kinase, which is quite rigid.

Induced fit model

The enzyme is not specifically made to fit, but is flexible, and changes the shape of the active site to allow the substrate to fit. It will return to its original shape after the substrate leaves. It is drawn similarly to lock and key, however the first enzyme is shown to be a different shape from the substrate. An example is the enzyme lysozyme, an antibacterial enzyme in human saliva, mucus and tears. The active site is a groove, allowing the sugars on the bacteria cell wall to fit in before closing around them. The enzyme then changes shape and hydrolyses the bonds holding the sugars together. This weakens the cell, causing the cell to absorb water by osmosis and burst.

Enzymes and activation energy

Activation energy is the minimum energy required for a reaction - to break the existing bonds and form new ones. High kinetic energy is needed for the molecules to interact, and higher kinetic energy gives a higher likelihood of the molecules collapsing. Enzymes work by lowering activation energy by modifying the shape of the substrate. This allows reactions to take place at lower temperatures, therefore requiring less kinetic energy.

Factors affecting enzyme action

Environmental factors, such as temperature and pH, can denature and permanently alter the shape of enzymes. This lowers the rate of reaction. Additionally, enzyme and substrate count can alter the rate of reaction. These factors are known as limiting factors.

Temperature

Increased temperature increases rate of reaction, as more kinetic energy is produced and particle collision possibility is heightened, rate doubling every 10 degrees rise in temperature. However, this reaches a limit for enzymes at around 40 degrees. At this temperature, the increased kinetic energy causes vibration to increase to the point is breaks the hydrogen bonds, denaturing the enzyme and altering the active site so the substrate cannot fit. This is a permanent change.

At low temperatures, the low kinetic energy causes enzymes to deactivate. However, this is not permanent, and they will return to normal function once the temperature rises.

pH

Most enzymes have an optimal pH. Small changes in this pH cause small reversible changes and reduce activity, however extremes can denature enzymes. Side chains on amino acids are affected by hydrogen or hydroxide ions. At a low pH, excess H+ ions neutralise negative charges. At a high pH, excess OH- ions neutralise positive charges. This disrupts the ionic and hydrogen bonds maintaining the shape of the active site, denaturing the enzyme.

Substrate concentration

With a consistent enzyme count, the rate of reaction rises with the substrate concentration. At low substrate levels, not all active sites are used, therefore they are not working to full capacity. However, there is a point where there is more substrate then active sites, with causes this rise to plateau.

Enzyme concentration

Only a small amount of enzymes are required for a large amount of reactions, due to their high turnover rate and reusability. However, the more enzymes the more reactions can be completed, meaning the rate of reaction is increased.

Enzyme graph equations

Temperature coefficient/ Q10

• This is a measure of the rate of a reaction’s rate of change as a consequence of raising the temperature by 10 degrees.

• A general rule is, every 10 degrees rise in temperature, the rate of reaction doubles.

• The formula is (with x equal to the initial temperature):

  • Rate of reaction at (10 + x degrees) ÷ rate of reaction at x degrees

Product formation graphs

• These graphs will have time on the x axis, and production mass on one side.

• Percentage increase in mass = increase in mass (initial mass - final mass) ÷ initial mass x 100.

• Rate of production (mg min-1) = increase in mass (initial mass - final mass) (mg) ÷ time (mins).

Enzyme inhibition

This is the decrease in the reaction rate of an enzyme-controlled action caused by another molecule, called an inhibitor. This combines with an enzyme to prevent it from forming an enzyme-substrate complex.

Competitive inhibition

• Competitive inhibitors have a molecular shape complementary to the active site, making them similar to the substrate.

• This causes them to compete for the active site.

• An increase in the substrate concentration reduces the effect of these inhibitors, as more substrate increases their chances of successfully beating the inhibitor for the enzyme. However, this is only true if the inhibitor is reversible.

• The opposite is also true - an increase in inhibitors lowers the reaction rate. However the reaction rate will still increase, as the enzymes still only react to the substrate.

• Examples include a Krebs cycle reaction in the mitochondrial matrix.

Non-competitive inhibition

• These bind to the enzyme at an ‘allosteric site‘ - a site other than the active site, therefore they do not compete with the substrate.

• They affect the bonds of the substrate and therefore alter the overall shape, including the active site. They prevent the active site from forming to fit the enzyme.

• Inhibitor concentration decreases reaction rate, but substrate concentration does not increase reaction rate as enzymes are permanently altered and do not have to directly compete.

• Non-competitive inhibitors can also bind irreversibly and reversibly, irreversibly deactivating the enzyme.

• Examples include heavy metals, such as lead and arsenic, which can cause death as they are irreversible.

Feedback inhibition

• Feedback inhibition only occurs along metabolic pathways, where the start reactant is catalysed by multiple enzymes, forming multiple products.

• This works with the final product becoming a non-competitive inhibitor to the first enzyme in the chemical pathways. This is a reversible inhibitor.

• This is done to prevent overproduction of the product, and therefore control the reaction.

Enzyme immobilisation

• Immobilised enzymes are fixed in position, such as in sodium alginate beads, cellulose microfibrils and columns. This is unnatural, and only done through human intervention.

• Immobilised enzymes can prevent instability. Free enzymes are highly sensitive to pH and temperature, while trapped enzymes create microenvironments enabling these increases in temperature and pH. It can also prevent the shape change that denatures an enzyme.

• The products are also not contaminated with the enzyme, as they are held in place.

• The reactions are more economical, as more product is produced.

• The amount of enzymes and reactants can be controlled, allowing for total control of the reaction.

• Enzymes are easily reused without moving them.

• These are used in medicine and industry.

• However, there is a lower rate of reaction than free enzymes, as it takes time for an substrate to diffuse to the enzyme.

Biotechnology

Continuous flow technology

• This is when a reactant is continuously flowed through a glass tube containing alginate beads containing an enzyme. The product will then flow through the bottom.

• One example is lactose free milk, produced by pouring milk through lactase enzymes.

• Pectin is another example. Crushed fruit is poured through pectinase enzymes, breaking down the juice. This makes the juice clearer, improve taste and increase the yield. Often, acids and chelating agents are used to advance the enzyme effect.

Biosensors

• This is when immobilised enzymes are used to detect the concentration of molecules, for example glucose.

• The enzymes are attached to a semi-permeable membrane, to keep non-reactants out. It is also inert (non-reactive) to stop additional reactions.

• This works in five steps:

  • 1 - The reactants are allowed through the semipermeable membrane, in glucose detection this is glucose and oxygen.

  • 2 - Enzyme-substrate complexes are formed between glucose oxidise and glucose.

  • 3 - The product is made, gluconic acid and hydrogen peroxide.

  • 4 - This product is detected by the electrode, which converts the chemical energy to electrical.

  • 5 - A digital reading is given of the glucose concentration in the sample.