Enzyme Kinetic Lab
Ah Seung Chong
Molecular Biology CTW: Enzyme Kinetic
Dr. Cruz
07/22/2010
Enzyme kinetics
Introduction
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 …show more content…
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
Molecular Biology CTW: Enzyme Kinetic
Dr. Cruz
07/22/2010
Enzyme kinetics
Introduction
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 …show more content…
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