Enzyme Kinetics

Enzyme Kinetics - 1 Marc Pomi 106040098 11/11/2009...

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1 Marc Pomi 106040098 11/11/2009 Professor Johnson and Oatis Enzyme Kinetics: The Hydrolysis of Nitrophenyl Phosphate by Alkaline Phosphatase The standard parameters of Michaelis-Menten kinetics were established in an experiment that examined the hydrolysis of nitrophenyl phosphate (NPP) by Escherichia coli (E. coli) alkaline phosphatase at pH 10.6 and about 25 C. The parameters consisted of the maximum velocity V max of 6.916E-04 and the Michaelis constant K M of 6.75E-07 respectively. Also, the product of the reaction HPO 4 2- was found to be a competitive inhibitor with a constant value K I of 6 x 10 -4 . The variation of the molarities and volume used created a large difference in the reaction rates. I. Introduction An enzyme is a protein that acts as a catalyst which, speeds up the rate of the reaction without modifying the overall Gibbs-energy change. A substrate is a reactant (other than the catalyst) in a catalyzed reaction. An enzyme works by binding to the substrate
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2 temporarily to allow for the reaction to occur at the lower energy. An inhibitor is a substance that reduces the rate of a chemical reaction which, works by binding to the enzyme (1). Kimball states that the rate an enzyme works is depending on concentration of substrate, temperature, presence of inhibitors, and pH (2). The factors affecting the enzyme concentration however though was limited to substrate concentration and the presence of inhibitors keeping the temperature and pH constant. Also, a special type of inhibitor called a competitive inhibitor was used. A competitive inhibitor is an inhibitor in which it takes higher substrate concentrations to achieve the same reaction rate at infinite concentration. This is caused by when the inhibitor and the substrate compete for the same active binding site on the enzyme as opposed to a non competitive inhibitor which the inhibitor binds to another site on the enzyme making the enzyme less efficient (2). According to Johnson and Oatis, families of enzymes found in places such as the bacterium E. coli and mammalian intestine are alkaline phosphatases (3). These reactions occur everywhere due to the vast distribution of E. coli and mammals. Enzymes are generally not selective in speeding up the process of hydrolysis of many phosphate monoesters to phosphate and neutral molecules. A general formula is as follows: Equation 1: R-OPO 3 2- + H 2 O → R-OH + HPO 4 2- where “R” is any organic entity. These reactions are useful in laboratory settings for the removal of phosphate from DNA and biological uses in the transportation of nutrients across cell membranes. To understand these processes better, the kinetics of the reaction was studied under a variety of
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3 conditions. Enzyme Kinetics was examined by a spectrometer under several conditions to see the changes in light absorbencies as the reactions proceeded. II. Theory
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Enzyme Kinetics - 1 Marc Pomi 106040098 11/11/2009...

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