Substrate concentration
Increasing the substrate concentration will increase the rate of reaction, as there are more substrate molecules to bind with the enzyme’s active sites. However if the substrate concentration carries on increasing, there is a point where the enzyme is working at its maximum possible rate and so additional substrate molecules that are not bound to the enzyme will not affect the rate of reaction.
Enzyme concentration
An increase in enzyme molecules in a given volume increases the rate of reaction, as there are more active sites to aid reactions at one time. This is the case when there is an excess of substrate. When there is a limiting amount of substrate, increasing the enzyme to a higher concentration than the substrate, will have no effect on the rate of reaction.
Inhibitors
Inhibitors inhibit the function of enzymes. There are two types of inhibition, competitive and non-competitive. Competitive inhibitors have a similar shape to the substrate and can bind to the active site of the enzyme briefly preventing the substrate from binding to it. The function of the enzyme is only effected if the concentration of the inhibitors rises to a point where the chance of the collisions between the substrate and enzyme is significantly lowered. In non-competitive inhibition a molecule binds to part of the enzyme away from the active site and causes a conformational change in the active site of the enzyme, thereby inhibiting the binding of the appropriate substrate molecule.
Cofactors
Cofactors are the non-protein part of the enzyme. Some are essential for reactions. Cofactors influence enzyme function. Cofactors are not bonded to the enzyme.
CATALASE
In this experiment, the enzyme used is Catalase. Catalase is found in all living cells, it breaks down a poisonous chemical hydrogen peroxide (H2O2) into harmless products, oxygen and water:
Catalase has one of the highest turnover numbers for all known enzymes (40,000,000 molecules/second). This is an important characteristic for dealing with, for example the build up of hydrogen peroxide in the blood.
It is a large protein molecule made of a folded chain of hundreds of amino acids. There are four sub-units of Catalase each containing a haem group. The locationof activity of the enzyme is in the haem group. Thehaem group takes two electrons from two molecules of hydrogen peroxide, making two water molecules and one oxygen molecule.
2H2O2 2H2O + O2
3D structure of catalase from E. coli.
The structure was solved using X-ray crystallography ()
In catalase, haem functions as a prosthetic group. A prosthetic group is a tightly bound, specific non-polypeptide unit required for the biological function of some proteins.Haem consists of a protoporphyrin ring and a central iron (Fe) atom. A protoporphyrin ring is made up of four pyrrole rings linked by methene bridges. Four methyl, two vinyl, and two propionate side chains are attached. The iron can either be in the ferrous (Fe++) or the ferric (Fe+++) oxidation state.
Other enzymes also have variable effects on reactions dependent on the concentration. For example, carbonic anhydrase and maltase.
CARBONIC ANHYDRASE
Carbonic anhydrases are enzymes that catalyse the hydration of carbon dioxide and the dehydration of bicarbonate in the human body.
CO2 + H2O <-----> HCO3- + H+
Carbonic anhydrase isozymes are metalloenzymes consisting of a single polypeptide chain complexed to an atom of zinc. They are incredibly active catalysts, with a turnover rate (kcat) of about 106 reactions per second.
These carbonic anhydrase-driven reactions are of great importance in a number of tissues where the concentration of the enzyme will affect the rate of reaction. Examples include:
Parietal cells in the stomach secrete massive amounts of acid (i.e. hydrogen ions or protons) into the lumen and a corresponding amount of bicarbonate ion into blood.
Pancreatic duct cells do essentially the opposite, with bicarbonate as their main secretory product.
Secretion of hydrogen ions by the renal tubules is a critical mechanism for maintaining acid-base and fluid balance.
Carbon dioxide generated by metabolism in all cells is removed from the body by red blood cells that convert most of it to bicarbonate for transport, then back to carbon dioxide to be exhaled from the lungs.
MALTASE
Maltase is a glycosidase. It catalyses the hydrolysis of glycoside bonds.
The action of maltase on maltose is usually written as:
Increasing the concentration of maltase will affect the rate of production of glucose.
EXPERIMENT
Prediction:
As the surface area of the potato becomes larger, more enzyme molecules are released, increasing the enzyme concentration. A higher concentration of enzymes will increase the rate of reaction at constant temperature, pH and other limiting factors.
Preliminary preparations
The experiment was performed using a standard method given by a supervisor. The exercise was to highlight any potential difficulties and find ways to improve the method as well as noting any observations during the experiment. One problem that was found was the difficulty in cutting cubes from an irregularly shaped potato. The best solution was to cut all the sides off the potato, creating an even surface.
Inserting the cube of potato, pushing the bung into the test tube and starting the stopwatch at the same time was impractical. Having two people working together solved this problem.
Observations: When the potato was added to the catalase the solution bubbled. This indicated that is was reacting. The initial rate of reaction was slower than predicted and a small volume of oxygen was collected that was found to be difficult to measure accurately, but by three minutes there was a more measurable volume. This determined the necessary time period.
Diagram of Experiment
Apparatus:
5 clean test tubes- contamination may affect results
Bung and delivery tube
Tongs-if test tube is handled could effect temperature of catalase
2 clamp stands
2 clamps
2 bosses
Measuring cylinder- 50ml for collecting oxygen- chosen as small measurements will be made, a bigger measuring cylinder would give an insignificant displacement
Water trough
Water (enough to fill half of the water trough)
10 test tubes
Test tube rack
Stopwatch
Hydrogen peroxide (0.001M)
Measuring cylinder –50ml for measuring hydrogen peroxide
2 large potatoes
Ruler
Scalpel
Tile for cutting potato
Goggles
Apron – if available
Variables
The surface area of the potato
Controlled variables
The volume of hydrogen peroxide, the volume of water in a full measuring cylinder, the temperature (room temperature) and the pH. If any of these variables are changed this will affect the rate of reaction.
Risk assessment
Hydrogen peroxide is a toxic chemical it is advisable not to get it on your skin, if it does wash immediately with soap and water. When dealing with a toxic chemical running or eating in the lab is not recommended. Make sure a test tube rack is used to prevent test tubes from spilling. If there is a spillage wipe the surface with water. In case of a test tube breaking sweep up broken glass immediately and wrap in paper before disposing.
Method
- Set up the apparatus: clamps, bosses and clamp stands as shown in the diagram, with clamp B set about 15cm from bottom
- Cut sides of potato with scalpel on the cutting tile, to make a cube of potato
- Measure 5 cm along opposing sides and mark with scalpel
- Cut the width of the potato with scalpel using the ruler, along the marked points. Be sure that the cut is vertical
- Along the unmeasured side make a mark with scalpel at 1cm intervals
- Do the same on the opposite
- Cut vertically down along the corresponding marks with scalpel, using the ruler
- Lay each potato piece on its largest face
- Along the two unmeasured sides mark 1cm intervals with scalpel
- Cut vertically down along these marks with a scalpel using a ruler
- Stop when 5 potato cubes have been cut
- Refer to the calculation of surface area table showing the dimensions of each set of potato if needed
- Put one of the potato cubes to one side
- Place one of the cubes on the tile and measure 2 cm with a ruler from one end and mark with the scalpel
- Do the same on three faces that can be seen
- Cut through these marks with scalpel dividing the cube into two pieces, put to one side next to the selected uncut potato cube
- Place another potato cube on the tile and measure 1 and 2cm with a ruler from one end and mark with the scalpel
- Do the same on three faces
- Cut through these marks with scalpel dividing the cube into three pieces and place with the prepared cubes of potato
- Place a third potato cube on the tile and measure 1, 2 and 3cm with a ruler from one end and mark with the scalpel
- Do the same on three faces
- Cut through these marks with scalpel dividing the cube into four pieces and place with the three sets of potato cubes put to one side
- Place the final potato cube on the tile and measure 1,2,3 and 4cm with a ruler from one end and mark with the scalpel
- Do the same on three faces
- Cut through these marks with scalpel dividing the cube into five pieces and place with the rest of the potato cubes
- Half fill the water trough with water and place in front of clamp B
- Submerge the 50ml measuring cylinder into the water in the water trough
- Tip the measuring cylinder upwards slightly(under water) to release any trapped air bubbles
- Turn the measuring cylinder upside down keeping the top of the measuring cylinder in the water and attach to clamp B
- Attach test tube to clamp A
[**REPEAT EXPERIMENT FROM HERE]
-
Measure 20 cm3 of Hydrogen peroxide in a measuring cylinder into the test tube
- Place the bung and deliver tube into test tube to minimise the evaporation of the hydrogen peroxide
- Feed the delivery tube into the inverted measuring cylinder
- Place stopwatch in front of clamp A, ready for use
- Lift the bung off the test tube and drop the potato with the smallest surface area into the test tube
- Push the bung down and start the stopwatch at the same time
- Time for three minutes so that a measurable amount of oxygen is produced
- Take out delivery tube from the measuring cylinder
- Record the volume of oxygen collected/volume of water displaced in the results table and fill in the surface area of the potato, volume of hydrogen peroxide and time period
- Take the off the bung from the test tube and detach the test tube from the clamp and place in the test tube rack
- Take a clean test tube and attach to clamp A
- Tip away the hydrogen peroxide and dispose of potato cubes
- Repeat experiment from **asterisked point above. Follow the same method, using a different set of potato cubes with increased surface area.
- Repeat the whole method to get a second set of results and record in the repeat column on the same results table.
- Repeat the experiment if the results in both the initial and repeated experiments do not match the predicted results: the rate of reaction increases with surface area
Recording results
-
Draw a results table with the headings: surface area of potato(cm2), volume of hydrogen peroxide, time period, volume of oxygen collected (cm3), and volume of oxygen collected(cm3) (repeat experiment)
- Write an informative title for the results table
-
Draw another table of results with the headings surface area of potato(cm2), volume of Hydrogen peroxide, volume of oxygen collected (cm3/min), volume of oxygen collected(cm3/min); (repeat experiment) and average volume of oxygen collected(cm3/min)
- Write an informative title for the second results table
- Fill in the surface areas on each set of potato and the amount of Hydrogen peroxide on the second results table
-
Calculate the volume of oxygen collected in cm3/min for the first set of results: divide the volume of oxygen collected by the time period for each surface area
- Record on the second results table
-
Calculate the volume of oxygen collected in cm3/min for the repeated experiment
- Record on the second results table under the repeat column
-
Calculate the average of oxygen collected in cm3/min and record in the last column
Presentation of results
-
Draw a graph on graph paper, with Volume of oxygen collected(cm3/min) on the x axis and surface area on potato(cm2)
-
Plot the volumes of oxygen collected in (cm3/min) for each surface area from the second results table on the graph
- Draw a line of best fit with a red pen
-
On the same graph plot the volumes of oxygen collected in (cm3/min)form the repeat experiment of each surface area from the second results table on the graph
- Draw a line of best fit with a blue pen
- Draw a key on the graph: red line- experiment results and blue line-repeat
- Write an informative title for the graph
- Draw another graph with the same axis titles
-
Plot the average volumes of oxygen collected(cm3/min) for each surface area from the second results table on the graph
- Write an informative title for the graph
Bibliography
Biology 1 textbook by Mary Jones, Richard Fosbery and Dennis Taylor. Cambridge, 2000.
Biological Sciences review, volume 15, number 1. September 2002.
Introduction to advanced biology by C.J. Clegg. John Murray, 2000.
A-level and AS-level Biology by Alan Cornwell and Ruth Miller. Longman, 1990.
http://scholar.hw.ac.uk/site/biology/glossary.asp#8
http://crystal.uah.edu/~carter/enzyme/catalase.htm
http://arbl.cvmbs.colostate.edu/hbooks/molecules/carbonic_anhydrase.html
http://www.activescience-gsk.com/miniweb/content/enzymes/reaction.htm