Measuring enzyme-controlled reactions

Measuring enzyme-controlled reactions

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Enzymes are like biological helpers that speed up chemical reactions in living things. They don't make any permanent changes though. They just lower the amount of energy needed to start the reaction. Enzymes work both inside and outside of cells in living organisms. There are two kinds of enzyme-controlled reactions: anabolic and catabolic. Anabolic reactions help create bigger molecules, while catabolic reactions break down bigger molecules into smaller ones. In summary, enzymes are important because they help make chemical reactions happen faster and more efficiently in living things.


The activation energy is required with an enzyme and without an enzyme
The activation energy is required with an enzyme and without an enzyme

Enzyme activity is measured in katal using the International System for units (SI). However, that's too big to measure, so we use 1 μmol min-1 (which is around 16.67 katal) instead. When we round numbers, we usually round to significant figures or decimal places. For example, 0.0055834 to 2 decimal places is 0.01 and to 2 significant figures is 0.0056.

An enzyme unit is the amount of enzyme needed to transform1 μmol of substrate per minute under certain conditions. The specific activity of an enzyme is measured by the number of units per milligrams of protein catalysed.

To track enzyme-catalysed reactions, we can calculate the rate at which a product is made or the rate at which the substrate is used up.

Factors affecting enzyme-controlled reaction rates

Several factors can affect how fast a reaction happens, including:

Temperature: as temperature increases, enzyme activity usually increases too. But if the temperature gets too high, the enzyme can get damaged and stop working altogether.

pH: each enzyme works best at a different pH level. If the pH is too high or too low, the enzyme can become denatured and stop working.

Substrate concentration: when there's more substrate, there are more chances for the enzyme to collide with it and trigger the reaction. However, once all the enzyme active sites are occupied, the reaction rate can't increase any further.

Enzyme concentration: more enzyme usually means a faster reaction rate, up to a certain point. But if there's not enough substrate to go around, having more enzyme won't make a difference.

Inhibitors: these can block the active site of the enzyme, so the substrate can't bind. Or they can bind to an allosteric site, which changes the shape of the active site and makes it harder for the substrate to bind.

Enzyme activators: these are molecules that can bind to enzymes and make them work even better.

Effects of substrate concentration

Denaturation: the process by which a protein loses its shape (changes in 3D/tertiary structure) and will no longer bind substrate effectively. This is because the active site has been altered. Enzyme active site: a specific binding site for the substrate. Allosteric site: a region of the enzyme where a molecule can bind and alter the active site. You will find a more detailed explanation for the factors affecting enzyme activity in our Factors Affecting Enzyme Activity article.

Investigating enzyme-controlled reactions - required practicals

Let's have a look at some examples of practicals you may encounter.

The effect of temperature on the rate of reaction

In this practical, the aim is to investigate the rate of reaction of an enzyme-controlled reaction using a powdered milk suspension. The independent variable is temperature, and the dependent variable is the rate of reaction.

To conduct this experiment, you will need powdered milk suspension, trypsin solution (0.5%), distilled water, and hydrochloric acid (HCl) (0.1 M). You will use conical flasks, tubes, and other equipment provided by your teacher.

First, create two control samples by adding 5 cm3 of milk suspension to two tubes. Add 5 cm3 of distilled water to one tube to represent no enzyme activity, and add 5 cm3 of HCl to the other tube to represent enzyme activity.

Next, create three test samples by adding 5 cm3 of milk suspension to three test tubes. Place all the test tubes in a water bath at 10ºC for 5 minutes, then add 5 cm3 of trypsin to each and start the timer. Time how long it takes for the solution to become colourless. Repeat this process at different temperatures, such as 20ºC, 30ºC, and so on.

To record your data, use a table with three repeats for each temperature. Calculate the mean time taken for each sample to hydrolyse by adding the time and dividing by the number of tubes. Then, calculate the rate of reaction using the equation: Rate of reaction = 1 / mean time.

For example, if at 10ºC, the milk hydroly in50s, 55s, and 60s, the mean time is (50 + 55 + 60) / 3 = 55s. The rate of reaction for this temperature is calculated as 1 / 55s = 0.02 (2 d.p.) s-1. Plot the rates of reaction for each temperature on a graph to visualise the reaction rates.

In conclusion, this practical provides a method for investigating the effect of temperature on enzyme activity using trypsin and a powdered milk suspension. By measuring the rate of reaction at different temperatures, we can determine the optimum temperature for this reaction and better understand the factors that influence enzyme activity.

The effects of pH on the rate of reaction

Enzymes require a specific pH to work best. Enzymes have a narrow range, and the optimum activity will be at the highest rate. This practical will investigate the optimum pH for the amylase enzyme. Amylase is a catalyst for the hydrolysis of starch (amylose) to maltose. It is mostly present in plant tissue, which includes seeds. Starch will be broken down during seed germination to release glucose, a food source for the growing seed. Amylase works best at a neutral pH of 7 and a temperature of 37 ° C.

Amylase enzyme solution Starch solution pH solutionIodine solution

Set up the bunsen burner with the tripod over it - this is where you will place your beaker with the solution. Place the beaker containing water on the tripod above the bunsen burner and keep the temperature at 35 degrees (adjust the flame accordingly).Add a couple of drops of iodine solution onto the spotting tile. In the test tube - mix 2 cm3 of amylase, 2 cm3 of starch and 1 cm 3 of pH solution. Place the test tube in the beaker with water. Every 20 seconds pipette a few drops of solution on the spotting tile (containing iodine solution).Do this until the iodine solution no longer turns black, and note the time. You can repeat this with different pH solutions. Changing different independent variables, such as the pH or concentration of the enzyme or substrate, will allow you to investigate their effects on reaction rate. Another enzyme you could potentially use is pepsin. Pepsin is an enzyme that catalyses the breakdown of proteins. Pepsin is present in most animal stomachs, and unlike most enzymes, its optimum pH is very low. This means it thrives in acidic environments.

The effect of changing substrate concentration on rate of reaction

During cellular respiration, your mitochondria produce hydrogen peroxide (H202) as a by-product of oxygen combustion (substances react with oxygen to give off heat). Hydrogen peroxide is toxic to cells, so how does the body get rid of it to avoid harm? Several enzymes, including catalase, break down hydrogen peroxide. When it is broken down, it produces oxygen and water.In this practical, you will investigate how different concentrations of hydrogen peroxide affect oxygen production.Hydrogen peroxide at a range of concentrations (example concentrations can include: 1M, 0.8M, 0.6M, 0.4M, 0.2M)Potato which has been cubed (this will act as the source of catalase)Large bowl Measuring cylinderWeigh the potato pieces and place them in the conical flask filled with 5cm3 hydrogen peroxide. Fill a large bowl with water. Fill the measuring cylinder and place it upside down inside the large bowl (make sure that no air bubbles are present).The bung in the lid of the conical flask will have a tube extending out, which will go under the measuring cylinder. Record the volume of oxygen in the measuring cylinder at regular time intervals (e.g. every 10 seconds).Repeat with other hydrogen peroxide concentrations.Increasing the concentration of hydrogen peroxide, i.e., the substrate will increase the reaction rate. However, this will not go on forever (unless an unlimited amount of enzyme is available), so when all enzyme active sites are occupied, no more substrate can be converted.The effect of changing enzyme concentration on the initial rate of reactionAnother factor affecting the reaction rate is enzyme concentration. The more enzymes you have, the quicker the reaction rate until all active sites are occupied. In this practical, you will measure the absorbance using a colourimeter (measures light transmittance).Milk powder solution Trypsin solution (1%)Distilled waterDilute the stock trypsin solution to produce concentrations of 0.2, 0.4, 0.6 and 0.8 %.In the control solution, add 2cm3 of stock trypsin (1%) and 2cm3 of distilled water. This solution will be used to set the absorbance to zero. In your test solution, add 2cm3 of milk suspension and 2cm3 of stock trypsin (1%). Mix and place in the colourimeter to read the absorbance. At regular 15 second intervals, record the absorbance reading. Do this for 5 minutes. Now repeat this for the other trypsin concentrations.You can use the rate of reaction formula in the previous section to calculate the reaction rate.The initial reaction rate will be directly proportional to the concentration of the enzyme. If unlimited substrate was present as the enzyme concentration increases, this increase could continue forever!When you plot the enzyme concentration against the reaction rate with your result, the points may not totally fall in a straight line. However, in the perfect world, this is how it will look (Figure 3).


The initial rate of reaction with increasing enzyme concentration
The initial rate of reaction with increasing enzyme concentration

When there is a limitation in enzyme concentration, the reaction will initially increase until all enzymes are occupied. It will reach the plateau (a flat line at a specific reaction rate).

Enzyme Controlled Reactions - Key takeaways Enzymes are biological catalysts that increase the rate of reaction. The reaction rate is affected by factors including pH, temperature, enzyme and substrate concentrations. You can measure how each factor affects the reaction rate by setting up experiments and changing the independent variable (i.e. temperature, pH etc.).Many different enzymes speed up chemical reactions, including trypsin, catalase, and amylase. Some enzymes are involved in digestion, waste break down and other metabolic processes.

Measuring enzyme-controlled reactions

How do substrate concentration and ph affect enzyme-controlled reactions?

An increase in substrate concentration will increase the rate of reaction, then it will plateau. The plateau occurs when all the enzyme active sites are occupied. The optimum pH will be different for each enzyme. Any variation in the optimum pH will reduce the rate of reaction and can also denature enzymes. This is because the amount of H+ ions will affect the tertiary structure, and therefore the active site of the enzyme. 

How are enzymes tested in biology?

Enzymes can be tested using different experiments. For example, the effect of pH on enzymatic rate can be tested by placing enzymes in different concentrations of acid/alkaline solutions.

What can affect enzyme controlled reactions?

Enzyme controlled reactions can be controlled by factors such as: Temperature pH Substrate concentration Enzyme concentration Presence of inhibitors Presence of enzyme activators 

What factors affect digestive enzymes? 

Enzyme activity can be affected by factors such as temperature, pH, and enzyme concentration.

What are the two kinds of enzyme controlled reactions?

Two main kinds: anabolic and catabolic.

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