Cell Lab 4_Updated.pptx

BIOL 3120 - Cell Biology Lab Lab 4. Enzyme Kinetics Rubaia Tasmin Teaching Assistant 9/23/2024 Email: [email protected] 1  Overview serve and analyze the kinetics of an enzymatic reaction through defined variable me & [Enzyme]...

BIOL 3120 - Cell Biology Lab Lab 4. Enzyme Kinetics Rubaia Tasmin Teaching Assistant 9/23/2024 Email: [email protected] 1  Overview serve and analyze the kinetics of an enzymatic reaction through defined variable me & [Enzyme] sess the key parameters Km and Vmax for Alkaline Phosphatase through ouble-reciprocal plot (Lineweaver-Burk plot) erpret & discuss your observations (both the raw data and the resulting paramet 2 Introducti Enzymes: Proteins that act as biological catalysts on: - Lower the activation energy - Act on specific substrate Active site: surface of the enzyme that binds the substrate - Change in temperature, pH, cofactors, or effectors affects enzyme’s structural stability and catalytic activity https://image.slidesharecdn.com/enzymesb-170810141102/85/enzymes-bpharm-1-320.jpg? cb=1502374286 3 Characteristics of an enzyme Enzymes enable the energetically favorable reactions to go faster. Enzymes are highly efficient & specific. Figure 3–50 The reaction catalyzed by lysozyme. Molecular Biology of the Cell 6 edition 4  Events at the active site of lysozyme Fig 3 - 49 (Movie 3.9) 5 How do enzymes speed up chemical reactions?  All chemical reactions require energy - Activation Energy is the amount of energy needed for a reaction to occur. - Enzymes lower the activation energy required for a reaction. https://us-static.z-dn.net/files/d8c/13d8e81ddfad5cd6705087139df7e0b4.png 6  Enzyme Kinetics: The study of the rates of enzyme-catalyzed chemical reaction  Michaelis-Menten equation Lineweaver-Burk equation 7 Michaelis-Menten Curve https://pharmafactz.com/medicinal-chemistry-understanding-enzyme- Alkaline Phosphatase  Isolated from calf intestine  Mw of the enzyme = 140,000 Daltons  Zinc ions: required for the enzyme’s structural stability & catalytic activity  Magnesium ions: act as allosteric activators (bind to protein at a site other than the active site and affect the activity)  pH optimum : 8.0-10.5 Things to note for today’s experiment: Substrate : p-nitrophenyl phosphate Product : p-nitrophenol (bright yellow) 8  Today’s Lab Procedure  Effect of time and [Enzyme] on product formation: Set the spectrophotometer to 405 nm Obtain 5 cuvettes 2 ml “Substrate-Cofactor Stock” to each cuvette Add 1 ml of the following: Cuvette 1 ml of Number 1 H2O 2 0.5x enzyme 3 1.0x enzyme 4 1.5x enzyme 5 2.0x enzyme Use cuvette 1 as a blank before each series of readings - Reading 1: after 5 min of 37oC incubation - Reading 2: after 2.5 min (time elapsed 7.5 min) - Reading 3: after 2.5 min (time elapsed 10 min) - Reading 4: after 5 min (time elapsed 15 min) 9  Today’s Lab Procedure  Determining Vo at various [S]: Use 18 cuvettes Add water, stock #1, and stock #2 as indicated in the manual Add 1 ml Buffer/Effectors to each cuvette Keep the cuvettes in 37oC for 5 min Add 1 ml enzyme buffer to each of the odd-numbered cuvettes Add 1 ml enzyme to the even-numbered cuvettes Return cuvettes to 37oC incubator for 9 min Read at 405 nm  Note: Odd-numbered cuvettes are blanks for even-numbered cuvettes Velocity = A x 18 10 Lab report format: File name for your lab report submission- BIOL3120_Section_Lastname_Firstname_L abN​ 1. Introduction: Background/concepts & purpose(s) of the experiment. 2. Materials: All chemicals, along with concentration, and instruments used. 3. Methods: Brief method/protocol. 4. Results: Tables, graphs, calculations 5. Discussion/conclusion: Interpret your results. Discuss unexpected findings. What improvements can you make? What is your conclusion? What is the significance of the lab? 6. Lab manual questions & underlined questions Times New Roman, 12 pt, Single-spaced, 1" margins. Complete sentences and paragraphs. Correct grammar. # Send me the Word document by email: [email protected] 11 Lab Exam 1 is on Monday 9/1 (usual time & room). Lab Exam 1 covers labs 1 through 4. Please bring a basic calculator on the exam day. 12