Enzyme Kinetics: Inversion of Sucrose

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EXPERIMENT 24: ENZYME KINETICS INVERSION OF SUCROSE ABSTRACT Using polarimetry, the kinetics of the inversion of sucrose was investigated and the rates of the enzyme-catalyzed reaction were ascertained. Six standardization runs, three with the enzyme and three without the enzyme, were performed and analyzed in order to the determine the specific rotation of sucrose. The optical rotation angles of solutions containing various concentrations of the substrate were measured and the initial rates were calculated. Using the Michaelis-Menten mechanism, the rate constants k2cE(0) and Km were determined by construction of a Lineweaver-Burk plot and linear regression of the data. Initial rates of solutions of the same consistency were determined by measuring the optical rotation angles at various temperatures. The data obtained was used to construct an Arrhenius plot and the activation energy for the reaction was determined. The values obtained through this experiment are as follows: α(0)=4.42 ± 0.06˚, α∞=-1.355 ± 0.001˚, k2cE0= (3.00 ± 1.15) x 10-4 (M/s), Km=1.1 ± 0.4 x 10-1 (M), and k2=(3.60 ± 1.38) x 10-4(s-1). INTRODUCTION In the study of chemical kinetics, the rates of enzyme catalyzed reactions are a significant area of interest as virtually all biochemical reactions are catalyzed by this class of proteins.1 For enzyme-catalyzed reactions, the basic mechanism as shown below in equations 1 and 2, was first proposed by Michaelis and Menten and then verified by a study of the kinetics of the inversion of sucrose.1 Eqn 1: E+S ES Eqn 2: ES E+P In this simple reaction mechanism an enzyme, E, converts a substrate, S, into products, P, through the initial formation of an enzyme substrate complex, ES. Using

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