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Lab04-Enzyme_kinetics - BSCI 330 Laboratory Manual Spring...

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BSCI 330 Laboratory Manual Spring 2012 Carpenter et al. 2011. BSCI330 Laboratory Manual. University of Maryland, College Park. Page 1 LABORATORY EXERCISE 4: ENZYME KINETICS Introduction Enzymes are protein catalysts produced and utilized by all living organisms in cellular metabolic reactions. This includes both anabolic (biosynthetic) and catabolic (degradative) processes. Like any catalyst, whether organic or inorganic, an enzyme acts to greatly increase the rate of a specific reaction by lowering the activation energy barrier , while at the same time being neither consumed nor modified by the reaction itself (Appendix of Images: Figure 1). It is important to understand that an enzyme does not alter the thermodynamics of a given reaction, including equilibrium states; it merely alters the rate at which a reaction reaches equilibrium. This is important since most important metabolic reactions in cells would proceed far too slowly on their own to be compatible with life. The increase in rate provided by an enzyme can be remarkable some enzymes increase the velocity of the uncatalyzed reaction by a factor of a hundred million times! As for all proteins, the function of an enzyme is determined by its structure. The actual catalytic event occurs at the active site (Appendix: Figure 2), generally a small groove or notch that is determined by tertiary structure (very often the actual catalysis is carried out by a non-protein prosthetic group , either an organic coenzyme or an inorganic metal ion cofactor ). Therefore, enzymes are acutely sensitive to environmental conditions, such as pH, temperature or ionic strength, that can affect this delicate tertiary structure. Enzymes must be carefully controlled, so that metabolic reactions occur in just the right place and at just the right time, as uncontrolled activity of catabolic enzymes could actually result in the destruction of the cell or tissue by degrading the component macromolecules. Various control mechanisms are summarized in Figure 3 (Appendix of Images). Some methods of control employed by cells include covalent modification (phosphorylation or cleavage of an inactive precursor called a zymogen ), environmental conditions (pH), compartmentalization (contained in a separate membranous organelle such as a lysosome), and the production of inhibitors (factors that bind to the enzyme and alter its functional capacity). A given enzyme will in fact have multiple controls from several of these categories, creating a secure interlocking network. Trypsin In this laboratory exercise, you will work with the digestive enzyme, trypsin in several experiments to examine some of the kinetic characteristics of enzymes and enzyme-catalyzed reactions. You will also be asked to determine the possible mechanism of action of a naturally occurring inhibitor of trypsin.
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