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BC367L_4_2011 - BC 367 Experiment 4 Kinetic Properties of...

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BC 367 Experiment 4 Kinetic Properties of Acid Phosphatase Introduction Phosphatases are enzymes that remove phosphate groups from substrates. Phosphorylated compounds are widely distributed in living systems. They serve as storage forms for energy (e.g., ATP and phosphocreatine), as components of informational macromolecules (i.e., nucleotides and deoxynucleotides), as allosteric effectors of certain enzymes (e.g., fructose-1-6- bisphosphate), and as second messengers (e.g., cAMP, cGMP, inositol phosphates). Phosphorylation-dephosphorylation reactions of proteins, mediated by protein kinases and protein phosphatases, modulate many enzyme activities (phosphorylase, pyruvate dehydrogenase, etc.). It is therefore not surprising that phosphatases of many kinds can be extracted from many tissues. In general, these enzymes catalyze the hydrolysis of phosphate monoesters as follows: During this experiment, you will investigate the properties of an acid phosphatase from wheat germ. Acid phosphatases have optimal activity at pH values from 4 to 6, and are distinct from alkaline phosphatases with optimal activity in the pH range of 8-9. The function of the wheat germ phosphatase has not been definitively established, but it has been proposed that it is involved in the mobilization of the phosphate reserves of the seed. Most of the phosphate within a seed is esterified as inositol hexaphosphate (phytic acid). Since the free phosphate concentration in seeds is low enough to limit metabolic reactions, enzymes must be present to catalyze the hydrolysis of the phosphate reserve substances. During the first week, you will perform a kinetic study on wheat germ acid phosphatase to determine its K m and V max with p-nitrophenylphosphate as the substrate. You will also determine the nature of the inhibition of acid phosphatase by inorganic phosphate. For the second week, you will examine the X-ray structure of acid phosphatase in the Schupf lab and meet with your instructor to discuss your plans for the following week. During the third week, you will carry out a set of your own kinetic experiments to investigate the effect of reaction conditions or a potential inhibitor of your choice on the kinetic properties of wheat germ acid phosphatase. Approach Kinetics concerns the study of reaction rates and the conditions that affect reaction rates. One of the primary concerns of kinetics deals with the study of the changes in the rate of a reaction as a function of the reactant (substrate) concentrations. Such measurements can lead to the calculation of rate constants and, where appropriate, of equilibrium constants. Kinetics can be of great value for predicting or eliminating possible reaction mechanisms under consideration, since an observed reaction rate must satisfy the proposed mechanism.
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