Module 4 Lab Report/Enzyme Assays and Enzyme Kinetics

Background: In this module, Enzyme assays and kinetics were studied to understand the catalytic efficiency of binding of enzymes. Enzymes are biomolecules that act as catalysts in organisms to bring about specific biochemical reactions. As catalysts, enzymes do not act directly in the reaction, but perform necessary actions that bring about the reaction. This is often accomplished by causing conformational changes in biomolecules via influence on intermolecular forces (Hbonding, van der Waals forces, electrostatic bonds, and hydrophobic interactions). The analysis of these reactions can reveal information about the activity of the organism and biomolecules such as the enzyme kinetics. This module was a review of methods for analysis and quantification of enzymatic reactions. When handling enzymes one must follow rules such as controlling temperature with the use of ice, diluting only when necessary to allow stability, and avoiding abusive shaking or vortexing to avoid oxidation of amino side chains. 4A. The reactions included an irreversible reaction: p-nitrophenylphosphate  p-nitrophenol + Pi In this experiment, the substrate pNPP is a common substrate used for detecting alkaline phosphatase. When the reaction occurs, it absorbs light at 405 nm which is detected using a spectrophotometer. A reversible reaction: +¿ +¿↔acetaldehyde+NADH +H ¿ ethanol+NAD¿ The assay uses a spectrophotometer, a machine used to measure the amount of light a substance's absorbs, to combine kinetic measurements and Beer's law by calculating the appearance of product or disappearance of substrate concentrations. The spectrophotometric assay is simple, non-destructive, selective, and sensitive. For example, the NADH/NAD+ molecule is used in the enzymatic oxidation/reduction reaction. During these reactions NADH is often oxidized to NAD+ , or NAD+ is reduced to NADH. NADH absorbs light at 340 nm, however NAD+ does not hold that property. A spectrophotometer can be used to measure the change in absorbance of 340 nm light, thus indicating a change in amount of NADH. 4B. Using nPP in an acet