Enzymes: Rates of Reaction (A-level Biology)
Enzymes: Rates of Reaction
We have established that the tertiary structure of an enzyme determines the structure of its active site, and therefore its substrate binding ability.
We will now explore how factors such as pH and temperature can affect the tertiary structure of enzymes, and therefore impact rate of reaction.
Optimum Conditions for Enzymes
- Enzyme tertiary structures are sensitive to changes. Protein and enzyme tertiary structures are very sensitive to environmental changes and require optimal conditions to keep functioning.
- Changes in environmental conditions can cause a protein to denature. Denaturation means a protein loses its shape. The normal shape of a protein or enzyme is known as its native conformation. The reversal of denaturation is known as renaturation.
Changes in Temperature
Optimal temperature. As you increase temperature, the kinetic energy of reactants increases. This increases the rate in two ways:
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- More frequent collisions – the reactant particles move faster, collide more often with each other and with enzymes, so there are more successful collisions (leading to a reaction)
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- More successful collisions – the reactant particles have higher energy, so any given collision is more likely to be successful and result in a reaction.
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Denaturation. As you increase temperature further, bonds in the active site begin to break, and the tertiary structure is disrupted. This alters the specific shape of the active site, so it may no longer be complementary to the substrate. This can happen at low or high temperatures.
The Temperature Coefficient
The degree to which the rate of reaction changes with temperature can be calculated using the following equation:
where Q10 is the temperature coefficient. It represents how much the rate of reaction changes when temperature is increased by 10°C.
Let’s take this example scenario:
- The rate of reaction is 10 products produced per minute for a reaction occurring at 30°C.
- The rate of reaction is 5 products produced per minute for the same reaction occurring at 20°C.
Then Q10 for that reaction would be:
The value of Q10 = 2 tells us that the rate of reaction doubles when the temperature of the reaction is increased by 10°C. A Q10 of 3 would indicate the reaction triples with a 10°C increase, and a Q10 of 4 would indicate the reaction quadruples.
Enzyme-mediated reactions typically have a Q10 of 2.
Changes in pH
Optimal pH. Most proteins and enzymes optimally function at a natural pH of 7.4. However, certain proteins and enzymes can tolerate higher or lower pH levels. For example, the human protein, pepsin, which is found in the stomach, works best at a pH of 2, which is highly acidic.
Denaturation. Denaturation can occur at low or high pH. The enzyme is affected due to disruption of the ionic and hydrogen bonds in the tertiary structure, which leads to an alteration in the specific shape of the active site.
Substrate Concentration
Increasing [substrate] initially increases rate. Increasing the concentration of substrate will increase the rate of collisions, so there will be more successful collisions per second. This is assuming a constant enzyme concentration.
After a while the enzyme active sites are saturated. After a certain point, increasing the concentration of a substrate, while keeping the enzyme concentration constant, no longer increases the rate of reaction. We call this point the saturation point. The enzyme concentration is now the rate-limiting factor.
Enzyme Concentration
Increasing [enzyme] initially increases rate. Similar to increasing the concentration of substrate in a reaction, increasing the number of enzymes increases the rate by increasing the amount of collisions between enzymes and substrates.
After a while there is a lack of substrate. After a certain point, if the amount of substrate is kept constant, the rate of the reaction will not increase with increasing enzyme concentration.
The dotted line represents a reaction with unlimited substrate. If the supply of substrate is unlimited, addition of enzymes will continue to result in increased reaction rates.
Measuring Rate of Reaction
- Product formation or reactant consumption is used to measure rate. Rates of chemical reactions are measured by how quickly a reactant disappears or by how quickly a product appears.
- We can measure changes in concentrations during a reaction. Because enzymes take a substrate (reactant) and turn it into a new product, we can measure how enzymes affect reaction rates by taking samples from a chemical reaction at different time points and measuring the change in concentrations of our starting substrates and the final product.
Measuring rate of product formation
In the previous tutorial, we discussed how catalase is an intracellular enzyme which helps the breakdown of hydrogen peroxide into water and oxygen. To understand how quickly this reaction is taking place, we can measure the rate at which oxygen (i.e. a product) is being produced:
- Mix a solution containing hydrogen peroxide and catalase in a test tube.
- Seal it using a bung with a delivery tube.
- Place the end of the delivery tube into an inverted measuring cylinder filled with water.
- As oxygen is produced, it will displace the water in the measuring cylinder. You can then measure how much oxygen is being produced per minute.
Measuring rate of substance disappearance
Amylase is an enzyme which helps breakdown starch into maltose. From the previous tutorials, we know that we can test for the presence of starch using the Iodine test. Thus, by measuring when starch is no longer present in our sample solution (i.e. it has been completely broken down by amylase) we can calculate the rate of reaction between amylase and starch:
- Mix a solution containing starch and amylase in a test tube.
- Place a drop of Lugol’s solution in each well of a spotting tile.
- At each minute, use a pipette to take a sample from the starch/amylase mixture. Drop this into a well containing Lugol’s solution.
- The Lugol’s solution will turn blue-black if starch is present. Record the time when the solution no longer changes colour – this is when the reaction is complete. You can then use it to calculate the average rate of reaction (i.e. the rate at which starch is being broken down by amylase) per minute.
Measuring rate of reaction using a colorimeter
In tutorial 17 Tests for Carbohydrates, we discussed how colorimetry is a a quantitative method that allows the concentration of compounds in a coloured solution to be determined. We can also apply this method to enzyme-catalysed reactions that involve colour changes.
- Trypsin is a digestive enzyme which catalyses the breakdown of the protein casein in milk.
- As the reaction occurs, the colour of the milk gradually changes from white to clear. Light is able to pass more easily through the solution at the end of the reaction.
- The degree at which the light is able to pass through the solution throughout the duration of the reaction can be measured using a colorimeter. We can then use this measurement to calculate rate of reaction per minute.
Michaelis-Menten Kinetics
Enzyme-mediated reactions are most commonly modelled using the Michaelis-Menten equation. The equation relates the rate of reaction to the concentration of substrates present. It is expressed as follows:
- V0 – Initial Velocity of Reaction (moles/times)
- [S] – Substrate Concentration (molar)
- Vmax – Maximum Velocity of Reaction
- Km – Substrate Concentration at half Vmax
It can also be expressed in the form of a graph:
- Vmax represents the maximum velocity reached by the system, at maximum substrate concentrations. In other words, this is the maximum rate of reaction that can occur at saturation point.
- The Michaelis constant (Km) is the substrate concentration at half of Vmax. This graph can be used to identify the Michaelis constant, Km, which is the concentration of substrate needed for half of Vmax to be achieved.
- Km demonstrates an enzyme’s affinity for a substrate. Km is essentially a measure of how strongly an enzyme can bind to its substrate. It is an inverse measure i.e. the lower the Km, the greater the affinity. This is because if an enzyme can bind well to its substrate, you will need less of the substrate to achieve half Vmax.
- Km can be used to compare different enzymes. As Km is a constant, it is a useful value to compare the affinity different enzymes have for their substrates.
Immobilising Enzymes
- When enzymes are used commercially, they are often immobilised. Immobilised enzymes are enzymes bound to an inert, insoluble material, typically calcium alginate, resulting in gel beads or capsules.
- Immobilised enzymes have several advantages over mobilised enzymes:
- Easily separable: Usually, enzyme-mediated reactions occur in solutions and it can be extremely difficult to separate the enzyme and the product at the end of the reaction. Immobilising enzymes allow them to be easily separated from the product as they are attached to an insoluble material.
- Cheap: As immobilised enzymes can be easily removed from the reaction, it is much easier to reuse them, reducing the cost of whatever production process they are involved in.
- Resistant: Immobilised enzymes tend to have greater resistance to changes in temperature and pH compared to mobilised versions.
FAQs
Enzymes are proteins that act as biological catalysts, increasing the rate of chemical reactions in the body.
The tertiary structure of an enzyme refers to its three-dimensional shape, which is determined by the folding of the polypeptide chain that makes up the enzyme. This folding is influenced by the sequence of amino acids in the polypeptide chain, as well as by various interactions between different parts of the chain.
The tertiary structure of an enzyme is critical for its activity, as it determines the shape of the enzyme’s active site, which is where the substrate binds and the reaction occurs. The active site is typically a pocket or cleft in the enzyme’s structure, with a specific shape and chemical composition that allows it to interact with the substrate in a highly specific manner.
The folding of the polypeptide chain in an enzyme is stabilized by various types of interactions, including hydrogen bonds, van der Waals forces, disulfide bonds, and hydrophobic interactions. These interactions can occur between different amino acid residues in the same polypeptide chain, as well as between residues in different chains if the enzyme is composed of multiple subunits.
Overall, the tertiary structure of an enzyme is critical for its activity, as it determines the shape and chemical properties of the enzyme’s active site, and thus its ability to catalyze specific reactions.
The reaction rates for enzymes depend on various factors, such as temperature, pH, substrate concentration, and enzyme concentration.
Temperature: Enzymes have an optimal temperature at which they work most efficiently. At temperatures below the optimal temperature, the reaction rate will be slow, and at temperatures above the optimal temperature, the enzyme may become denatured and lose its activity.
pH: Enzymes also have an optimal pH at which they work best. Changes in pH can affect the shape and activity of the enzyme, leading to changes in the reaction rate.
Substrate concentration: The reaction rate increases with increasing substrate concentration until a point is reached where all enzyme active sites are occupied, and the reaction rate levels off.
Enzyme concentration: The reaction rate increases with increasing enzyme concentration until a point is reached where all substrate molecules are bound to enzyme active sites, and the reaction rate levels off.
In general, enzymes catalyze chemical reactions by lowering the activation energy required for the reaction to occur. They do this by binding to the substrate and stabilizing the transition state, making it easier for the reaction to proceed. The rate of enzyme-catalyzed reactions can be measured by monitoring the appearance or disappearance of the substrate or product over time.
Enzymes speed up the rate of chemical reactions by lowering the activation energy required for the reaction to occur.
Activation energy is the energy required for reactant molecules to reach a transition state, from which they can proceed to form products. Enzymes work by binding to the reactant molecules, or substrates, and lowering the activation energy required for the reaction to proceed. This allows the reaction to occur more quickly and efficiently than it would without the enzyme.
The binding of the substrate to the enzyme causes a conformational change in the enzyme, which brings the substrate into the optimal orientation for the reaction to occur. The enzyme may also provide a microenvironment that is conducive to the reaction, such as a specific pH or ionic environment.
Enzymes are highly specific in their interactions with substrates, meaning that each enzyme can only catalyze a specific reaction or set of reactions. This specificity is due to the shape of the enzyme’s active site, which is complementary to the shape of the substrate. When the substrate binds to the active site, it induces a conformational change in the enzyme that stabilizes the transition state and lowers the activation energy.
Overall, enzymes increase the rate of chemical reactions by providing an alternative reaction pathway with lower activation energy, allowing reactions to occur more quickly and efficiently than they would without the enzyme.
The active site is the region of the enzyme molecule where the substrate binds and the reaction takes place.
The shape of the active site is specific to a certain substrate, allowing only that substrate to fit and react. If the active site is altered or damaged, it may affect the rate of reaction as the substrate may no longer fit properly.
The rate of an enzymatic reaction can be influenced by various factors, but here are three key factors that have a significant impact on the rate of enzymatic reactions:
Substrate concentration: The rate of an enzymatic reaction increases as the substrate concentration increases, up to a point where all enzyme active sites are occupied. Beyond this point, the rate of the reaction will plateau, as all the enzyme molecules are already bound to substrate molecules.
Temperature: Enzymatic reactions have an optimal temperature at which they work most efficiently. At temperatures below the optimal temperature, the reaction rate will be slow, and at temperatures above the optimal temperature, the enzyme may become denatured and lose its activity.
pH: Enzymes also have an optimal pH at which they work best. Changes in pH can affect the shape and activity of the enzyme, leading to changes in the reaction rate.
Other factors that can influence the rate of enzymatic reactions include the concentration of enzyme, the presence of inhibitors or activators, and the ionic strength of the solution. It is important to note that different enzymes have different optimal conditions for their activity, and these conditions can vary widely depending on the enzyme and the specific reaction being catalyzed.
Increasing temperature increases the kinetic energy of the enzymes and substrate molecules, leading to a higher rate of reaction. However, very high temperatures can denature the enzymes and alter their shape, leading to a decrease in activity.
The optimal pH for an enzyme-catalysed reaction is specific to each enzyme. A change in pH outside of this range can alter the shape of the enzyme and affect its activity, leading to a decrease in the rate of reaction.
Increasing substrate concentration increases the rate of reaction up to a point, where all the active sites on the enzyme are occupied by substrate. Beyond this point, the rate of reaction remains constant, as the enzyme is saturated with substrate.
The optimum conditions for enzymes vary depending on the specific enzyme and the reaction it catalyzes. However, there are some general conditions that are known to be optimal for most enzymes:
Temperature: Most enzymes have an optimal temperature at which they work most efficiently. This temperature is usually around 37°C for enzymes found in humans and other warm-blooded animals, but can vary widely depending on the organism and the specific enzyme. Generally, the rate of the enzymatic reaction increases with increasing temperature until a point where the enzyme becomes denatured and loses its activity.
pH: Enzymes also have an optimal pH at which they work best. This pH is determined by the ionizable groups on the enzyme’s active site and can vary widely depending on the specific enzyme. For example, pepsin, an enzyme that breaks down proteins in the stomach, has an optimal pH of around 2, while trypsin, which works in the small intestine, has an optimal pH of around 8.
Salt concentration: Some enzymes require a certain salt concentration to function optimally. This is because the presence of salt ions can affect the charges on the enzyme and substrate molecules, and thereby affect the enzyme-substrate interactions.
Cofactors: Some enzymes require cofactors, such as metal ions or vitamins, to function optimally. These cofactors can help to stabilize the enzyme’s structure or participate in the reaction mechanism.
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