Abstract: The abstract is the last part of the paper written. It is a concise, compelling summary of your work such that a reader can decide whether the paper is worth reading. Often only abstracts are readily available and getting the full text requires additional effort and/or expense. Introduction • The introduction should begin with the relevant biology, physiology and commercial applications for lactase, and end with its pertinent biochemistry (MW, number of amino acids, secondary structure, quaternary structure, reaction catalyzed) Materials and Methods: No Materials and Methods Required Results Two sections to the Results for this lab: • • • Characterization of Lactase catalysis of the formation of o-nitrophenol from ONPG o Figure: Michaelis-Menten plot o In the legend, your determined values for Km, Vmax Characterization of the inhibition by galactose. o Figure: Michaelis-Menten plots of all uninhibited and inhibited experiments. o Figure: Lineweaver-Burk plots comparing uninhibited and inhibited by galactose experiments o In the legend: ▪ Km and Vmax value for the enzyme inhibited by galactose. ▪ Identify the type of inhibitor (competitive, uncompetitive) The results section should have professional quality figures and paragraphs summaries of the uninhibited and inhibited results. Discussion 1. Look up the structure of lactose and create a figure, labeling glucose, galactose and the glycosidic bond. 2. Look up the reaction catalyzed by lactase when ONPG is the substrate analogue. Create a figure showing the structure of ortho-Nitrophenyl-β-galactoside (ONPG) and of the products. a. In the figure legend, consider the structure of galactose and predict the sort of interactions would you expect to find between galactose and the enzyme in the active site? 3. Download and open the PyMol file, Lactase480.pse 4. Press “F1”. The view stored is a surface representation of lactase with lactose shown as sticks. Galactose is shown with yellow carbons and glucose with pink carbons? Create a figure from this image and address the questions below in the legend. a. Based on what you see, why would ortho-Nitrophenyl-β-galactoside (galactose as the sugar) be a good substrate analogue and ortho-Nitrophenyl-β-glucoside (glucose as the sugar) be a poor substrate analogue? b. Similarly, why would we expect galactose to inhibit the reaction with ONPG while glucose did not? 5. Press F2. The view stored is a close-up of the active site showing interactions between galactose and the enzyme. Waters are shown as red spheres and a sodium ion as a purple sphere. Create a figure from this image and address the questions below in the legend. a. Trp 518 does not have polar contacts with galactose. What might the role of Trp 518 be in binding? b. Asn 110 and Glu 393 do not have direct interactions with galactose. Explain how they indirectly interact with the galactose. c. Seven amino acids have direct interactions with the galactose. Identify the type of interaction for each. Some may be ambiguous. If you claim ambiguity, explain why. d. What two amino acids are near the sodium ion? What is their charge and what might their role be? MicroL Substrate 200 250 400 600 100 50 10 Slope inhibited Slope uninhibited 0.001968 0.003972 0.00154 0.008607 0.003176 0.008411 0.004349 0.006419 0.001188 0.00259 0.0004599 0.005496 0.0001063 0.0005981 Slope v Substrate 0.01 Slope 0.008 0.006 0.004 0.002 0 0 100 200 300 Substrate ul Slope inhibited Enzyme concentration - 1.914 mg/ml Linear (Slope inhibited) Slope vs Substrate 0.01 0.009 0.008 0.007 0.006 Slope ope v Substrate y = 9E-06x + 0.0031 R² = 0.4107 0.005 0.004 0.003 y = 7E-06x + 0.0002 R² = 0.9665 0.002 0.001 400 500 600 Substrate ul Slope uninhibited Linear (Slope uninhibited) 700 0 0 100 200 300 400 Substrate Vol (MicroLiters) s Substrate Inhibited series 500 600 700 F1 F2 F1 F2 F1 F2 F1 F2 Lactase UNDERSTANDING THE EXPERIMENT AND ANALYSIS Prep Stage DETERMINING APPROPRIATE EXPERIMENTAL CONDITIONS Prep Stage  The purpose of the prep stage is to determine conditions (enzyme, substrate, and inhibitor concentrations) that will result in a meaningful experiment  Experimental design is to vary a single parameter (e.g. enzyme concentration) to achieve a specific result  Examples:   Find an enzyme concentration that results in ΔAbs = 0.1/min  Determine the approximate Km  Determine the inhibitor concentration that results in ~50% inhibition Questions to ask:  Why is determining this parameter important for a good experiment?  What would happen if a value far from the desired value were chosen? Experimental Stage COLLECT DATA UNDER CONDITIONS THAT SUPPORT ANALYSIS Experimental Stage  In the Experimental Stage a single parameter is varied (e.g. substrate concentration) to determine its effect on the measured value  Example:   Vary substrate concentration and measure its effect on initial velocity Questions to ask:  How will the values chosen impact downstream analysis (e.g. using only high concentrations of substrate will result in V = Vmax)  How can the values chosen best support downstream analysis? Data Processing Stage PERFORM MATHEMATICAL OPERATIONS TO TRANSFORM RAW DATA INTO FORMATS SUITABLE FOR ANALYSIS Data Processing Stage  Experimental Data often requires manipulation to prepare for analysis  Examples:   Transform Experimental Units into Units suitable for analysis  Volume of Substrate added to [Substrate]  Δabs/sec to Δ[Product]/sec Questions to ask:  What is the desired data format for my processed data?  What units should my processed data be in?  What is the relationship between raw data format and the format desired for analysis? Analysis Stage PROCESSED DATA IS ANALYZED AND RESULTS OBTAINED Analysis Stage  Processed Data is analyzed/modeled and results obtained  Examples:   Determine Km and Vmax  Michaelis/Menten Plot (V vs [S]) and Solver  Lineweaver-Burk Plot (1/v vs 1/[S]) Questions to ask:  What is the mathematical relationship between the data and the parameters being modeled?  In the case of regression analysis, what is being varied and what is the criteria to determine model quality? Conclusion Stage RESULTS ARE CONSIDERED AND CONCLUSIONS DRAWN Conclusion Stage  Results are compared and conclusions drawn  Examples:   Internal Comparisons  Uninhibited and inhibited Km and Vmax values are compared to determine type of inhibition  Uninhibited and inhibited Km and Vmax values are compared to determine α and α’ values External Comparison  Results are compared with literature values Additional Info INFORMATION COMPILED FROM TEXTBOOK AND LAB DOCUMENTS Additional Info  kcat  Turnover number  For an enzyme with one active site per molecule, the turnover number is the number of substrate molecules transformed to product by one enzyme molecule per unit time.  Mathematically  kcat = Vmax / [Etot]  The enzyme concentration, [Etot],can be determined from the A280 value of the stock solution (see the “Determine Enzyme” tab) and the extinction coefficient for tyrosinase). Don’t forget the protein stock was diluted for the experiment Additional Info Relationship between Apparent and Actual Km and Vmax Values for Different types of Inhibitors Inhibitor type Apparent Vmax Apparent Km None Vmax Km Competitive Vmax αKm Uncompetitive Vmax/α' Km/α′ Mixed Vmax/α' αKm/α′ Additional Info  Ki  Dependent on inhibitor type  Competitive   Uncompetitive   α = 1 + ([Inhibitor] / Ki) α' = 1 + ([Inhibitor] / Ki) Mixed  α = 1 + ([Inhibitor] / Ki)  α' = 1 + ([Inhibitor] / Ki)
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