Molecular Biology CTW: Enzyme Kinetic
Enzymes are biological catalysts or assistants, without enzyme many of important processes of life could not happen. Most of enzymes are proteins that help speed up chemical reactions by lowering amount of activation energy needed for the reaction1. Enzymes are usually highly selective, only bind to specific substrate and convert it to product at a particular rate1. The rate of the reaction affects by substrate concentration: the more the substrate presents, the faster the reaction2. It also can be influenced by affinity of enzyme to substrate: the higher the affinity of enzyme to substrate, the faster the reaction2. In this experiment, Pyruvate Kinase, an enzyme that catalyzes the final metabolic reaction in glycolysis which converts phosphoenolpyruvate to pyruvate, was used to analyze the effect of substrate concentration on velocity of reaction and effect of inhibitory and activators which effects the affinity of enzyme to substrate on reaction rate. The rate of reaction determined by the amount of product formed, pyruvate, over time through OD510nm reading. Thus, the standard curve for pyruvate was first constructed to establish the correlation with OD510nm and pyruvate concentration. The standard curved was used throughout the experiment to analyze the reaction rate of enzymes in different environments. Second part of the experiment was performed to see the effect of substrate concentration on velocity of the reaction. Different level of ADP or substrate was used and corresponding OD510nm reading was measured. Both Michaelis-Menton Plot and Lineweaver-Burk plot were constructed to determine maximum velocity, Vmax and Michaelis constant, Km. Vmax is a maximum rate at which the enzyme can perform the reaction and Km is an inverse measure of the affinity or strength of binding between the enzyme and its substrate2. The last part of the experiment was performed to analyze the effect of inhibitor and activator proteins on reaction rate. The input of different effectors such as KCl, KNO3, NaCl, ATP, F-1,6-BP were used for different samples. The result of last part of the experiment was compared with different groups in the class. For the result, the low Km and Vmax value was expected because very low volume of pyruvate kinase was used. Therefore, concentration of pyruvate will be low overall. For part C, the effectors such as KCl, KNO3, and NaCl will increase the velocity of reaction because they are chemical ions which will help enzyme to bind to substrate by increase its affinity. F-1,6-BP is another big protein molecule which will inhibit pyruvate kinase by fighting for same active site, thus it will decrease the reaction rate. ATP is the product of the pyruvate kinase reaction, thus reverse reaction might occur and it will slow down the reaction rate. Material and Method
The first part of the experiment started from construction of standard curve for pyruvate. Total of 1.5ml of different volumn of pyruvate and deionized water added to twelve test, two test tube for each standard, to make following final pyruvate concentrations: 0M, 15M, 30M, 60M, 90M, 120M. 1.5 ml of Dinitrophenylhydrazine added to the twelve test tubes. Dinitrophenylhydrazine allowed pyruvate to be detected spectrophotometrically at 510nm. The test tubes were incubated at 30°C in heat block to trigger enzyme –substrate reaction and then 7ml of NaOH added to the test tubes to halt the enzyme reactions. OD at 510nm was measured and the recorded data was used to construct the standard curve. For the second part of the experiment which was performed to see the effect of substrate concentration on velocity of the reaction. Each 0.1ml of PEP, 0.1ml of MgSO4, and 0.2ml of Pyruvate Kinase (addition of pyruvate kinase followed after addition of ADP) were added to ten test tubes, two test tubes for each standard, different amount of substrate, ADP...