Enzyme Kinetics Measuring enzyme rates of reaction using the Spectrophotometer/ SpectroVis Probe Objectives: 1. Define enzymes and describe the activity of enzymes in cells. 2. Differentiate between competitive and noncompetitive inhibition. 3. Discuss the effects of varying different environmental conditions such as pH and temperature on the rate of enzyme activity. 4. Discuss the effects of varying enzyme and substrate concentrations on the rate of enzyme activity. 5. Discuss the scientific process, propose hypotheses, and make predictions based on you hypotheses 6. Practice communication of scientific results by constructing and interpreting graphs of enzyme activity. Introduction: Living cells perform a multitude of chemical reactions very rapidly because of the participation of enzymes. Enzymes are organic catalysts that speed up chemical reactions without being used up or altered in the reactions. The material with which the enzyme reacts, called a substrate, is modified during the reaction to form a new product. Because the enzyme emerges unchanged and ready to bind with another substrate, a relatively small amount of enzyme can be used with a huge amount of substrate. The active site of the enzyme will bind with its specific substrate, forming an enzy,e-substrate complex. It is here, at the active site, that the catalysis takes place. When it is completed, the complex dissociates into reusable enzyme and product(s). Enzymes are proteins (with perhaps a cofactor added) and are highly specific. Because they lower the activation energy needed for reactions to take place, they accelerate the reactions. They do NOT determine the direction the reaction will go towards equilibrium.
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Enzyme activity is influenced by many factors. Environmental factors such as pH and temperature, may change the three dimensional shape of the enzyme and alter its rate of activity. Specific chemicals may bind to the enzyme to modify its shape. Chemicals that MUST bind to the enzyme to make it more active are called activators. Cofactors are non protein substances that bind to the enzyme and are required for normal enzyme activity. Organic cofactors are called coenzymes. Chemicals that shut off enzyme activity are called inhibitors and may be competitive or noncompetitive.
Figure 1. Enzyme activity. A substrate or substrates bind to the active site of the enzyme forming the enzyme-substrate complex, which then dissociates into enzyme and product(s). The enzyme may catalyze the addition or removal of a molecule or a portion of a molecule from the substrate to produce the product (a), or the enzyme may catalyze the splitting of a substrate into its component subunits (b)
There are two ways of measuring enzyme activity. (1) Determine the RATE of disappearance of the substrate and (2) determine the RATE of appearance of the product. Background:
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As you know, most cells are too small to observe with the unaided human eye, therefore we rely upon the microscope to assist us in observing individual cells. But when we wish to ask questions about functions inside the cell, we need some tool for separating the parts of a cell from each other. From you first year biology course, you should remember that plant cells consist of dense cell walls, starch grains, chloroplasts, mitochondria, a nucleus and cytoplasm in which you will find many enzymes. The parts of a cell can be released by homogenization in a blender. The released cellular components can be separated by their different densities in a centrifugal force field. Mrs. R will blend potato tissue, pour the homogenate into test tubes and spin them in centrifuge. The high speed will cause the densest particles to sediment as a pellet at the bottom of the test tube. Lighter particles will sediment in bands above this bottom layer. Dissolved molecules do not sediment at the centrifuge speeds we will use, but instead remain in the supernatant liquid. If we wanted to study a particular enzyme found in potato cells (which IS what we are going to do), that enzyme would be in the supernatant fluid.
Enzymes are a class of proteins that serve as biological catalysts, that is, they speed up (by at least 1,000,000 times) the rates of chemical reactions in living cells. An enzyme catalyzes a reaction by binding to a substrate molecule and altering its molecular structure so that the substrate is more readily converted to a different molecule, or product. After releasing the product molecule, the enzyme is then available to be used again. Because only one or a few different kinds of substrate molecules are able to bind to the surface of a give enzyme, each enzyme typically catalyzes only one or a few different chemical reactions. Thus, most cells contain several thousand different kinds of enzymes in order tos carry out all of the chemical reactions necessary for life. In todays laboratory experiment you will study some basic properties of enzyme-catalyzed reaction using the potato-cell enzyme polyphenoloxidase (also called catecholase) is an enzyme found in a wide variety of plants and is responsible for the darkening of cut surfaces of many fruits and vegetables. Polyphenoloxidase catalyzes the oxidation of catechol to orthoquinone. Catechol, oxygen, polyphenoloxiase, and ortho-quinone are colorless, but ortho-quinone undergoes a series of changes to form a red product. catecholase CATECHOL
+ ½ Oxygen
→
ortho-quinone + Water → red product
Let’s follow the formation of product by observing the increase in red/brown coloration Why do plants carry out a reaction like this? Apparently, the above reaction occurs as a response to injury; small amounts of catechol are present in the cell vacuoles of man plant tissues, while the enzyme is present in the cell cytosol (i.e cytoplasm). When the tissues are damages, the catechol is released and the enzyme converts it to ortho-quinone, which is a natural antiseptic. The browning on fruit is a protection against disease. Polyphenolo0oxidase is found in humans and other animals, where it goes by another name, tyroinase, and is involved in poroducing the skin pigment, melanin. Preparation of Enzyme (Catecholase) 1. Wash, peel and weigh potato and place in a Waring blender. Add 2.5 times the volume of water as compared with the potato. 2. Blend for two minutes on high speed. Filter mixture through coffee filters. 3. Centrifuge and collect the supernatant for ten minutes. Keep on ice.
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5. Supernatant contains the catecholase.
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4. Decant (pour off) supernatant from centrifuge tubes into a beaker or flask and keep in a container of ice water.
Introduction to Part I This exercise will investigate the results of catechol oxianse ativity. In the presence of oxygen, catechol oxidase catalyzes the removal of electrons and hydrogens from catechol, a substance found in plants. Catechol is converted to benzoquinone, a pigment product. The hydrogens combine with oxygen forming water. The pigment products are responsible for the darkening of fruits and vegetables, such as apples and potatoes, after exposure to air.
In this exercise, you will use an extract of potato to test for the presence of catechol oxidase and to establish the appearance of the products when the reaction takes place. Hypothesis Before making your hypothesis, review the information given about the activity of catechol oxidase and construct a hypothesis for its presence or absence in potato extract. Remember that the hypothesis must be testable. Prediction Predict the result of the experiment based on your hypothesis. To test for the presence or absence of catechol oxidase in potato extract, your prediction would be what you e expect to observe as the result of this experiment. Part I – The Baseline – All teams must perform this part of the lab first. Into a clean cuvette prepare a Blank as follows: 1 ml 0.3 M Catechol 1ml 0.1 M Buffer (6.8pH)
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Blank your spec 20 at 540 nm using the above mixture prior to adding the enzyme.
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3.25 ml D water
Prepare a Baseline Mixture by duplicating the above cuvette (USE ONLY 3 ml D water). At time = 0, add 0.25 ml of potato extract and MIX THOROUGHLY. Cover cuvette with parafilm and invert to mix. Record the obsorbance at 540 nm at 30-second intervals. Remove the tube in between readings to prevent overheating. Save tube for later comparisons. The baseline mixture has what four ingredients? 1. 2. 3. 4. Data Table I: Time (30 second intervals) for about 7 minutes + Absorbance (OD) Part II – Student-developed lab with different variables. Having completed Part I of the lab, you and your team learned the basic technique that can be used to determine the rate of reactions between an enzyme and its substrate. Different variables can be tested to determine their effect on the RATE of enzymesubstrate interaction. Your task will be to select one of the variables listed below and develop a protocol to test your hypothesis. Possible Variables: Concentration o Substrate Concentration of Enzyme Temperature of Reaction mixture pH of Reaction mixture
Task:
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Another choice
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Effect of reaction inhibitors on rate of reaction
1. Research background (bibliography required) 2. Develop a hypothesis (testable) 3. Make a Prediction 4. Develop a protocol that takes into account dependent and independent variables, control, range of variable, number of samples (or trials), what data you will collect? 5. Analyze you data 6. Present your findings to the class (numerical and graphical) and determine if your data supports or does not support your hypothesis.
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7. You will have three days to develop your experiment, collect data, put together your presentation to the class.