Chemical reactions will only occur when the reacting molecules collide during random molecular movement. The speed of the reaction depends on the number of collisions. This in turn depends on the amount of random molecular movement and the concentrations of the molecules. The conditions within living organisms do not always favor a speed of reaction fast enough for the body's requirements. It is necessary for the body to produce and use substances called enzymes. Enzymes are biological catalysts which alter the rate of chemical reactions without affecting the products formed. An enzyme works by bringing together (surface catalyst) the reacting substances, the substrates, so that they are more likely to react and form the product. An enzyme does not actually take part, but acts as a surface on which the reaction can occur. An enzyme does not make a chemical reaction occur that doesn't happen anyway, but it changes the rate at which the reaction occurs. This simple enzyme animation will demonstrate all of the aforementioned properties.
Enzymes are proteins composed of long chains of amino acids. Each has a precisely defined composition with a complex structure. The nature of the active site on any particular type of enzyme varies according to specific protein structure. Any one enzyme can only conformationally (shape) match certain substrates and so can only catalyze certain reactions.
Use Enzyme #4 for these practice exercises
Although thousands of different enzymes are known, the same general approach may be used for the study of any of them. The speed of any enzyme controlled reaction can be measured either by the disappearance of the substrate or by the appearance of the product. See graph and answer the questions below:
Figure 1-Course of an Enzyme Catalyzed Reaction
1. Describe the course of the enzyme catalyzed reaction shown in the above Figure 1.
2. What conditions would change the shape of the curve?
It is only at the moment when the reaction starts that all of the factors are controlled. From then on substrate concentration falls, back reaction or inhibition may begin and some enzyme may even be destroyed, as proteins are very sensitive to to high temperature and extreme pH. However, in practice, measurement over a period of a few minutes will usually give a progress curve that is straight enough for its average slope to be a good approximation to the initial slope. Then in order to obtain the initial velocity of the reaction divide the amount of substrate consumed or product formed by the time, i.e. y/x.
The initial velocities of reaction can be obtained under different conditions and compared to find out the characteristics of an enzyme. The pH, substrate concentration, enzyme concentration, incubation time and temperature can be varied.
Usually only one parameter is varied at a time, the rest being kept constant at what are thought to be suitable values to obtain meaningful results. Because this kind of study is concerned with the rates of reaction-and hence the idea of movement-it is termed enzyme "kinetics" from the Greek word meaning "move".
A thorough investigation in the laboratory of an enzyme catalyzed reaction is time consuming. However, so that you can have experience in planning investigations, drawing conclusions from the results and using your conclusions as the basis of new investigations, the computer program ENZKIN simulates a series of enzyme catalyzed reactions and gives the results as soon as you have put in the conditions of the reactions.
The simulation allows you to choose, for any of six enzymes, one varying factor. You state the range over which it varies and set values for the other factors. For example you may want to set values for pH, temperature and starting volumes of substrate and enzyme and consider the product formed over time intervals varying between 0 and 60 minutes. Similarly you may prefer to maintain a constant incubation time, but vary another factor such as temperature over a certain range. In this way the program ENZKIN allows you to perform quickly and easily a range of investigations on the six enzymes.
Operating Instructions:Chart of Keywords
Keyword Function START For the guided version of the program ENZYME To choose an enzyme PH To set the pH TEMP To set the temperature VOLUME VOLUMES
To set both the enzyme and substrate volume ENZVOL To set the enzyme volume SUBVOL To set the substrate volume TIME To set the incubation time VARY To select the varying factor (variable) GO To begin the calculation and display the results EXPLAIN For an explanation of the program HELP For the list of keywords FINISH To end the program
When first using ENZKIN it is best to type the keyword START when Option? appears on the screen. This will provide you with a guided version of the program. An enzyme and a suitable pH is automatically selected and time set as the varying factor. You are asked to set the time range, volumes of solutions used and the temperature.
The minimum and maximum possible values for the range of any of the factors and the minimum possible interval between them are shown in the table below:
Factor Minimum value Maximum value Minimum interval pH 0 14 1 Substrate volume/cc 0 9 1 Enzyme volume/cc 0 9 1 Incubation time/min 0 60 10 Temperature/C 100 10
In the simulation the test tube always has at least 1cc of buffer solution in it. The total volume in which the reaction takes place is 10cc, the capacity of the test tube. If the volume you provide exceeds 9cc you will be asked to restate new volumes. If the total volumes are less than 9cc, extra water is added to the test tube.
Values are displayed in the table at the top of the screen. When all values have been entered the computer program will calculate the amount of product at eleven, about equally spaced time intervals within your chosen range.
After the results have been plotted, Option? appears again at the top of the screen. You can change any of the conditions set by typing the appropriate keyword from the Chart of Keywords above, or type HELP in the ENZKIN program and you will get a list of Keywords. When you have entered the new value type GO to obtain the results. Particularly after altering the varying factor it is important to check in the table at the top of the screen that you have provided values for all factors before typing GO.
To become familiar with using the program, try the following investigation:
Answer the questions below that deal with the little investigation you just performed:
1. How do your computer generated progress curves compare with the progress curve in Section B, Figure 1 above? Are they the same or different? Explain.
2. Why is the product concentration unchanged in the last 10 minutes of the first reaction?
3. What differences in the results are there between the first and second reactions?
Enzymes are sensitive to pH. For every enzyme there is an optimum pH at which it functions most effectively and for most there is only a restricted range of pH in which they will work at all. For this reason the pH must be maintained at certain levels within the body for the correct functioning of particular enzymes.
The existence of optimum pH is a consequence of the acidic and basic groups present in enzymes and other proteins.
1. What is the optimum pH of the enzyme investigated?
2. What is the enzyme activity at the optimum pH?
When you know the optimum pH for any enzyme you can investigate the relationship between product concentration and time by running the program at different temperatures.
Enzymes like other proteins are affected by extreme temperature changes. The protein structure on which activity of the enzyme depends becomes irreversibly changed at high temperatures and the protein is said to be denatured. At low temperatures the structure may become temporarily distorted by the formation of ice crystals in certain parts of the molecule. In either case if the structure of the active site is altered the enzyme will no longer be able to bind to the substrates and catalyze reactions.
Try using the program to investigate:
a. a temperature you consider very suitable for life.b. a temperature 15 to 20 degrees lower.
c. a temperature 15 to 20 degrees higher.
Each run should be at the optimum pH for the enzyme and use 1cc of enzyme solution, a large substrate volume and vary the time between 0 and 60 minutes. To change the temperature but maintain all of the other values, type the keyword TEMP after Option? appears on the screen. State the new temperature, then type GO for another set of values. Record the results you obtain from each run.
1. What is the relationship between the product concentration and the time at the three temperatures?
2. Suggest two possible reasons why the progress curves are not straight lines?
3. Why do the progress curves have different initial slopes?
In the previous investigations both the enzyme and substrate concentrations were kept constant. However, they are factors that may vary in nature and the concentrations available for the enzyme catalyzed reaction may affect the course of the reaction.
1. What factors could vary the concentrations of substrates in nature?
2. Why are there variations in the amounts of enzyme available for reactions?
Use the computer program to investigate the effect of varying substrate concentrations. Choose a small enzyme volume and select an appropriate pH and temperature. The enzyme volume can be set independently by using the keyword ENZVOL.
1. At what initial substrate concentration is the most product formed?
2. Is more product formed when the initial concentration of substrate in increased?
Now vary the enzyme concentration. Choose a small substrate volume and select a suitable pH and temperature for the enzyme. The substrate volume can be set independently by using the keyword SUBVOL.
1. How does the enzyme concentration affect the amount of product and the speed at which it is formed?
The initial substrate concentration will determine the amount of product that can be formed, but the rate at which this occurs will depend on the amount of substrate compared to enzyme since the enzyme molecules provide the 'active sites' for the reaction to occur. The more enzyme molecules present the more substrate molecules can react at any one time. In addition enzymes have different speeds of action. This is known as the turnover number, that is the number of substrate molecules that one molecule of enzyme turns into product per minute. This varies from a few to several million, depending on the enzyme. The enzyme catalase, that was demonstrated in the lab, is found in the liver and has one of the higher turnover numbers known. It can 'process' almost six million substrate molecules in one minute. Its action is seen when catalase reacts with its substrate hydrogen peroxide and oxygen is released.
After years of hard study and laboratory work you've finally reached your goal of researcher for one of the five best biotech companies in the country. Enzkin is the most profitable biotech stock in the field of enzyme research. The completion of the Human Genome Project has the companies researching proteomics scrambling to investigate the gene products resulting from the informatics data.
Further References:a. Leading Edge-The Human Proteomeb. MIT Technology Review-Proteomics
Enzkin has been taking a different tact from other companies and is seeking to enter the industrial processing market. The genomes of nonhuman species are being deciphered using the techniques developed by genetics researchers and your company is looking at enzymes that have been engineered from bacterial informatics data.
The genome of the archeabacterium Thermatoga has been completed (Karl Stetter) and your company has access to the data base. Thermatoga lives in temperatures that are at the boiling point of water. It is a bacterium thought to be the earliest of the life forms on the planet and it has moved center stage in the search for the ancestors of life on earth.
Because this organism can both tolerate extreme conditions of temperature and continue to metabolize normally, it must contain proteins that are heat resistant. The primary structure of the proteins and the globular folding produced by the order of the amino acids confers heat resistant properties to what has traditionally been thought to be extremely labile (heat sensitive) molecules. Many of these proteins are heat tolerant enzymes and would be valuable commodities in industrial processing that involves high temperature reactions.
Five enzymes have been engineered as the result of the cracking of the Thermatoga genome. Your job is to determine the optimum conditions at which these enzymes operate. The enzymes' value in industry is dependent on these conditions and your work is critical in keeping Enzkin on top of the biotech market.
Your research group will be assigned one of the five enzymes and your task is to determine the optimum temperature and pH at which your molecule can produce 9000 micromoles of product from its specific substrate. Technicians hired by Enzkin have run hundreds of tests and collected data on the effect of pH, temperature, and the concentration of enzyme and substrate on the micromole output of product for each of the five enzymes in question. This information has been placed in a data base with a program that will allow you to manipulate the data in simulated experiments in order to determine each enzyme's optimal operating conditions.
Here's your problem defined:
What are the optimum pH and temperature for enzyme number _____that will produce 9000 micromoles of product in the shortest amount of time?
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*For security reasons, you will not be given either the name of the enzyme or the substrate on which it works. The company wants you to determine the physical conditions affecting the enzyme's operation.
As a group you need to determine these optimum conditions in the least number of steps. You must manipulate the variables until you think you have found the points of reference. Let the chief researcher know immediately when these points have been determined as he will then test you to assure the completion of your task.
All members of the group must be able to explain the entire process used to determine the end points. The chief researcher will ask each member of the group to perform at least one of the steps in the process of determining optimum temperature and pH in the shortest period of time. The awarding of grades will be based on the explanations and the number of steps it takes the group to arrive at a correct answer.
5 steps with explanations = 1006 steps with explanations = 95
7 steps with explanations = 85
8 steps with explanations = 75
9 steps with explanations = 65
An extra 5 points will be given to groups that finish the project correctly in one class period.
Good luck!