Marcos, Nelissa S.
Institute of Chemistry, University of the Philippines, Diliman, Quezon City 1101 Philippines
The rationale of the experiment is basically founded in the concept of reaction rates as affected by enzyme, and how the enzyme works is competed by a competitive inhibitor, thereby impeding the forward reaction. In this experiment, o-diphenol oxidase, an enzyme that causes the browning in fruits, was extracted from banana and reaction rate of this was established with various concentrations of catechol, the substrate, using the Michaelis-Menten, Lineweaver-Burk, Hanes-Woolf and Eadie-Hofstee plots. The plots were generated using the slope of absorbance readings against time plots. Absorbance can be used to detect reaction rate as this notes color intensity signaling product formation. The inhibition of o-diphenol oxidase by p-hydroxybenzoic acid is a competitive inhibitor, however, results in the experiment were not conclusive enough to determine that. Km values should increase if there is competitive inhibition, however, in this experiment, Km decreased in the four plots. Introduction
Enzymes are protein catalysts that hasten the rate of a chemical reaction but are recovered fully at the end of the process. The mechanism follows temporary binding of the enzyme to the substrate and, as a result, lowering the activation energy needed to convert the substrate to product.
The rate at which an enzyme works is influenced by several factors. One of these is the concentration of substrate molecules for the higher the amount, the faster the enzyme molecules collide and bind with them. The concentration of substrate is represented as [S] and is expressed in molarity. Temperature is also considered because as it rises, molecular motion causing collisions between enzyme and substrate speed up. But as enzymes are proteins, there comes a point when the enzyme becomes denatured and ineffective. Presence of inhibitors affects reaction rate of enzymes and there are different types of inhibitors depending on its affinity to a particular binding site: competitive inhibitors are molecules that bind to the same site as the substrate, thereby preventing the substrate from binding but are not changed by the enzyme, and noncompetitive inhibitors are molecules that bind to some other site on the enzyme reducing its catalytic power. The conformation of a protein is also influenced by pH and as enzyme activity depends greatly on its conformation, its activity is affected just the same.
The study of the enzyme works rate is called enzyme kinetics. Enzyme kinetics, in this experiment, is treated as a function of the concentration of substrate available to the enzyme.
Figure 1. Michaelis- Menten plot (Vi vs. [S]).a
Figure 1 shows the plot of Vi as a function of [S]. At low values of [S], the initial velocity, Vi, increases almost linearly with increasing [S]. But as [S] rises, the increments in Vi level off (forming a rectangular hyperbola). The asymptote represents the maximum velocity of the reaction, designated Vmax. The substrate concentration that produces a Vi that is one-half of Vmax is designated the Michaelis-Menten constant, Km (named after the scientists who developed the study of enzyme kinetics). Km is approximately an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the Km, the greater the affinity and so the lower the concentration of substrate needed to achieve a given rate. Figure 2. Lineweaver-Burk plot (1/Vi vs. 1/[S]).a
Figure 2 shows that plotting the reciprocals of the same data points yields a "double-reciprocal" or Lineweaver-Burk plot. This provides a more precise way to determine Vmax and Km. Vmax is determined by the point where the line crosses the 1/Vi = 0 axis so the [S] is infinite. The magnitude represented by the data points in the plot decrease from lower left to upper right. Also, Km equals Vmax...
References: a) http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/EnzymeKinetics.html
b) Hanes, CS (1932)
c) Dowd, JE; and Riggs, DS (1965). "A Comparison of Estimates of Michaelis–Menten Kinetic Constants from Various Linear Transformations". Journal of Biological Chemistry 240
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