3#1 Enzyme Kinetics Objectives: • To gain an understanding of how enzymes function in biological reactions. • By the end of this lab, you should be able to generate and interpret a standard curve and a Hanes-Woolf plot. • By the end of this lab, you should be able to define competitive and noncompetitive inhibition and you should be able to determine which type of inhibition applies when given experimental data like those you will generate during this lab. Pre-lab Preparation • Read this lab manual chapter • Read Chapter 8 of your textbook on “An Introduction to Metabolism”, especially pages 152-156 on enzymes. • Review Chapter 2 of the Lab Manual on “Lab Skills” for help with pipeting, dilutions, and using the spectrophotometer . Post-lab Assignment : • Complete the Post-lab assignment posted to the course Canvas site. This assignment is due at the start of lab next week . Background: Enzyme-Catalyzed Reactions Chemical reactions are processes that somehow change the structure of a substance or substances during which reactant(s) are converted to product(s) . Reactants do not suddenly become products, but instead their structures follow some kind of a path in order to undergo this conversion. Along this “path,” the molecule(s) require an input of energy to get through certain states. Think of a road that transverses a mountain range: in order to get from one side of the mountain range (the reactant state) to the other (the product state), a car must put in energy to go over the mountains. The amount of energy that it takes to go over this energy “hump” is termed the activation energy of a reaction. Enzymes are organic molecules (typically proteins) that catalyze chemical reactions by lowering the activation energy required to go to product (changing the “path” the reaction takes to one that requires less energy). Again, you can think of the car going over the mountain analogy. It is very difficult for a car to go over the top of a mountain, but it is much easier for it to go through the mountainside, by using a tunnel. This speeds up the rate of the reaction for a given set of conditions. In fact, if enzymes were not present in cells, most of the chemical reactions would not proceed at measurable rates at physiological temperatures (e.g. temperatures of living systems). The enzyme catalyzes the reaction by creating an intermediate compound called
3#2 the enzyme–substrate complex. Each enzyme has at least one active site where the substrate(s) binds to the enzyme. At the active site, the substrate(s) is held rigidly in the optimal orientation for chemical interactions to occur between the substrate(s) and the enzyme, thereby lowering the energy needed for the reaction to occur.
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