Factors That Affect Enzyme Action
Factors That Affect Enzyme Action
Objective
This unit aims to understand different factors and how they affect the rate of reaction.
Factors
Different factors can affect enzyme action. This includes:
1. Temperature
2. pH
3. Substrate concentration
4. Enzyme concentration
5. Inhibitor concentration
Mechanism
When enzymes are added to the solution of substrate they collide with each other. With time substrate concentration decreases thus, no collision also decreases. This results in a decrease in the rate of reaction.
Thus, the rate is fastest at the start of reaction because the numbers of substrates are maximum.
Temperature
When temperature increases à no of effective collision à increases rate of reaction increases.
At optimal temperature enzyme activity is maximum.
After this, the H bonds holding enzyme molecule together start breaking. Thus, the enzyme denatures.
This distorts the shape of the active site of enzyme à decrease enzyme-substrate collision à rate of reaction decreases.
Most of the enzymes work at an optimal temperature of 37degree C.
Effect Of pH
Enzymes maintain their normal structure at a very narrow range of pH. This is called an optimal pH.
Optimal pH:
Optimal pH is the pH at which enzymes activity is maximum.
Most enzymes work at an optimal pH of 7-7.45.
After this, the bonds holding enzyme molecules start breaking. Thus, enzyme activity decreases.
Effect Of Enzyme Concentration:
When there are more substrates than enzyme limiting factor is enzyme concentration:
Increasing enzyme concentration increases the chances of effective collision thus the rate of reaction increases.
But, increasing enzyme concentration after a certain point does not affect the rate of reaction. Now, the substrate concentration is less.
Effect Of Substrate Concentration:
When there are more enzymes than substrate:
Limiting factor is substrate concentration:
Increasing substrate concentrate à increases the rate of collision à thus the rate of reaction is increased.
But the rate will increase up to a certain point then there will be no effect on the rate of reaction because now enzymes are more than the substrate.
Effect Of Inhibitor Concentration
Inhibitors
Inhibitors are molecules that inhibit enzyme action.
Competitive Inhibitors
They have the same shape as the substrate.
Binds with the enzyme at the active site thus, preventing the substrate from it.
Increasing the concentration of the substrate can reverse inhibition.
Non Competitve Inhibitors
They do not have the same shape as the substrate.
Binds with the enzyme at a site other than active site and changes the shape of the enzyme and its active site.
Increasing concentration of the substrate cannot reverse inhibition.
Investigating Factors Affecting Enzyme Action
Temperature
For investigating the effect of temperature any reaction can be used. Here we are using Hydrogen peroxide reaction in the presence of catalase.
H2O2à H2+O2 (catalase)
Procedure
• Take conical flasks with H2O2 solution in them and place them in a water bath. Each water bath should be set at a particular temperature.
• Take a set of test tubes and add a catalase solution in them.
• Place these test tubes in the same water bath.
• Wait for the flask and test tube to come to the right temperature. Check with a thermometer.
• Take out the first flask after it has come to the correct temperature.
• Place it on a sensitive top pan balance.
• Add a catalase solution to it and immediately note the initial reading.
• Take readings every 30 seconds for about 3 minutes. Readings will decrease as oxygen is given off.
• Repeat the same with other flask placed at other temperatures.
• Calculate the initial rate of reaction for each flask. Then work out the gradient of the graph for the first 30 seconds.
Investigating Effect Of pH
To investigate effect of pH on enzyme action, we can use the same reaction that we used for temperature.
H2O2 à H2+O2 (catalase)
• Keep enzyme concentration, temperature and substrate concentration the same for all tubes.
• Vary the pH by adding different buffer solution to each tube containing catalase.
• Plot the graph with the readings you obtained.
Investigating Enzyme Concentration
The same reaction will be used here as we have used in the above methods.
H2O2 à H2+O2 (catalase)
• Prepare a catalase solution as we have done before.
• Make different dilutions of this solution.
• Take solution in different test tubes and add 1 cm3 of distilled water to the 2nd tube. Then keep increasing the amount of water added by 1cm3 for the rest of the tubes.
• Add solution to tube fitted with a gas syringe. Mark these tubes with waterproof markers.
• Place these tubes in a water bath set at 30 degrees C.
• Take another set of test tubes. Add 10 cm3 of H2O2 to each tube. Note the concentration of H202 should be the same.
• Place these test tubes in the same water bath and leave them for 5 minutes.
• Wait for all tubes to come to the right temperature.
• Take one tube of H202 and add it to one of the enzyme solutions. Note the amount of gas produced after 2 minutes.
• Repeat the same for other tubes as well.
• Plot your result on a graph.
Investigating Effect Of Concentration Of Substrate
It can be done in the same way as we did for enzyme concentration.
Note: Keep the enzyme concentration the same this time and vary the concentration of H202.
Michaelis Menton Equation
VO=VMAX[S]/KM[S]
It relates the rate of enzymatic reaction with the concentration of substrate present.
Here,
Vmax is the maximum rate of reaction that can be achieved at maximum substrate concentration.
Km is called as Michaelis constant. It is the inverse of affinity. Thus, it is the measure of affinity or enzyme-substrate binding. If km is low affinity is high that is less amount of substrate is required to reach the maximum rate.
Immobilizing An Enzyme In Alginate
Enzymes have a lot of commercial importance as well. They are used in industries on a large scale to speed up chemical reactions.
The problem is it is very expensive to use a new enzyme every time. For that reason, most industrialists prefer reusing the same enzymes.
But, it is very difficult to separate the enzyme product complex due to its small size. That is why enzymes are immobilized for reuse.
Immobilizing enzymes means they are attached to an inert substance such as calcium alginate. Thus, during the reaction enzyme keeps attached to sodium alginate and do not mix with the product. Then it is easily removed from the product.
Method
One way how an enzyme can be immobilized is to drop the mixture of enzyme and sodium alginate on CaCl2. Sodium alginate when the drop on cacl2 turns into solid with enzymes inside.