08 Kinetics - B iochemis ry E duca ion Department of...

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Unformatted text preview: B iochemis ry E duca ion Department of iochemistry & Molecular iology University of New Mexico BIOC 423 Int oducto y Biochemist y Enzyme Kinetics OBJECTIVES • Describe how enzyme reactions can be measured • Describe how the Michaelis-Menten assumptions limits the use of kinetic data • Discuss the significance of Km, Vmax and Kcat measurements • Be able to calculate Km, Vmax and Kcat values from kinetic data • Be able to use kinetic data to answer enzyme related questions • Using kinetic data be able to distinguish between competitive and non- competitive inhibitors OUTLINE Definitions Enzyme Activity Specific Activity Velocity Michaelis-Menton Kinetic Analysis Graphing Enzyme Reactions Michaelis-Menten Assumptions [S] >> [E] [ES] = constant = amount of E added [P] = 0 [EP] is ignored Lineweaver-Burk Plot Enzyme Inhibition Competitive Noncompetitive LECTURE The measurement of enzyme reactions provides an important tool that is used in forensic science, analytical chemistry, biomedical science and health care research and patient care decisions. In addition, the pharmaceutical industry develops compounds which interfere with enzyme reactions and other ligand binding reactions. Consequently, the measurement of enzyme kinetics is an extremely important tool that is used in several laboratories and disciplines of the life sciences. It is not a topic that can be avoided in any biochemistry survey course. Following the course of an enzyme reaction As discussed earlier in the course, enzymes catalyze the change in a substrate without itself being consumed by the reaction and results in the formation of a product. This reaction, in its simplest form, can be described by the following reaction. S + E ↔ P + E In the equation, S refers to the substrate, E refers to the enzyme and the product of the reaction is indicated with the letter P. In order to evaluate the progress of the above reaction it is necessary to determine either rate at which substrate disappears ( Δ [S]/ Δ t) or the rate of product appearance ( Δ [P]/ Δ t). In both cases these are measurements of a velocity. The units for this velocity term are generally in mMol/min. The exact method of measuring the reaction is obviously dependent upon the enzyme that is being measures. The reactions must be set up so that either the substrate (S) or the product (P) can be easily detected. There are multiple methods to accomplish this measurement. Direct measurement: The enzyme reaction can be measured directly if there is a measurable property of either the substrate or product that can be followed as a function of time. For example, if there is a color generated or lost during the reaction the enzyme can be measured by spectrophotometry. If there is a change in viscosity during the course of the reaction, then the physical property of viscosity can be used to follow the reaction course....
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This note was uploaded on 04/05/2008 for the course BIOCHEM 423 taught by Professor Osgood during the Spring '08 term at New Mexico.

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08 Kinetics - B iochemis ry E duca ion Department of...

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