During the early stages of an enzyme purification protocol, when cells have been lysed butcytosolic components have not been separated, the reaction velocity-versus-substrateconcentration is sigmoidal. As you continue to purify the enzyme, the curve shifts to theright. Explain your results.This is an allosteric enzyme and you must use a Lineweaver-Burk plot to determine KM and Vmaxcorrectly.This is an enzyme that displays Michaelis-Menten kinetics and you purify away an inhibitor.This is an allosteric enzyme and during purification you purify away an activator.This is an allosteric enzyme displaying a double-displacement mechanism and during purification youpurify away one of the substrates:This is an enzyme that displays Michaelis-Menten kinetics, and you must use a Lineweaver-Burkplot to determine KM and Vmax correctly.

If a reaction velocity v/s substrate concentration curve shifts to the right, then the curve becomes…

Answered: During the early stages of an enzyme…

Biochemistry

9th Edition

ISBN:9781319114671

Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Chapter1: Biochemistry: An Evolving Science

Section: Chapter Questions

Problem 1P

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Assume that the experiments performed in the absence of inhibitors were conducted by adding 5 μL of a 2 mg/mL enzyme stock solution to an assay mixture with a total volume of 1 mL. Take into account that XYZase is a monomeric enzyme with a molecular mass of 45,000 Daltons. Hint: To calculate the ???? in units of per second (s−1), you must first determine the ???? in micromoles per second (μmol/sec). Please explain step by step
There are six classes of enzymes, namely oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. (i) (ii) Cell disruption is one of the steps performed during enzyme production. Explain the reason of performing cell disruption in enzyme production and provide THREE (3) enzyme extraction methods for this type of enzyme. Explain the principles of gel electrophoresis as one of the methods used for enzyme purification.
200 ml of a 2% protein solution are available, containing an enzyme to be purified. Half of the sample is subjected to method A, consisting of fractionated precipitations, and 5 ml of final solution are obtained, with a protein concentration equal to 5 mg / ml and enzymatic activity equal to 2000 U / ml. The other half is subjected to method B, consisting of ion exchange chromatography, and a final solution of 10 ml is obtained, with a protein richness equal to 10 mg / ml, and with an enzymatic activity equal to 2000 U / ml. You want to know: a) Which of the methods has provided the purest enzyme. b) By which of the methods has the greatest amount of protein been obtained.
The Lineweaver-Burke plot was originally developed in order to "linearize" the data obtained from enzyme kinetics experiments, in order to facilitate the determination of kinetic parameters. Why is it not considered to be an accurate method for this purpose? It is very difficult to draw a straight line on a computer. It is very difficult to calculate the variables required for the "x" and "y" axis. It is more accurate to use the standard "V versus [S]" plot to determine Vmax and KM- The plot weights the least accurate data points the most heavily. It is no longer considered to be acceptable to extrapolate from known data.
You have performed protein purification on your new favorite enzyme using a protocol which involves the following steps/samples: crude extract, ammonium sulfate cut, ion exchange and gel filtration. You need to run 25ug of protein from your crude extract sample on an SDS-PAGE gel. You have determined that the protein concentration of your crude extract sample is 2.1mg/ml. Your total sample volume is 30ul. You have water for your diluent and 6x SDS-loading dye to prepare your sample. List the components of your prepared sample. Note: you will only have access to a P20 and a P200 to prepare this sample.
You are interested in purifying an enzyme X and decide to use an affinity chromatography followed by an enzymatic assay. You obtain the following data: Total volume of [protein] the sample (ml) (mg/mL) (U) |Total activity Sample name Specific activity |(U/mg) |Affinity | chromatography 2 0.12 5000 20833 You have obtained a 10X fold purification and a yield of 10% at the Affinity step. What was the protein concentration of your crude lysate sample, if its volume was 15 mL? Detail your answer by showing the calculations, and do not forget the UNITS!
A purified protein sample was used in a reaction, resulting in an activity of 696.7 nmol min-1. The reaction volume was 145.0 µL and the final volume before loading the plate was 1,050 µL. The total reaction time was 4.25 min. The amount of protein used in the reaction was 4.270 µg. Calculate the specific activity of the sample (in nmol min-1 µg-1).
Lisa decides to obtain values for the Km and Vmax of an enzyme that was isolated from liver cells.. Using the Michaelis Menten plot. In what kind of measurements are needed and what would be needed to plot on a graph to estimate Km and Vmax?
From a kinetics experiment, the Vmax was determined to be 450µM∙min-1. For the kinetic assay, 0.1mL of a 0.05mg/mL solution of enzyme was used, and the enzyme has a molecular weight of 125,000 g/mole. Assume a reaction volume of 700µL. Calculate the kcat (in sec-1) for the enzyme.
Based on the Lineweaver-Burke plot attached. Kinetic data were generated in the (1) absence of any inhibitor, (2) presence of 15 µM of a reversible inhibitor, or (3) presence of 20 µM of a second (distinct) reversible inhibitor. Purified enzyme concentration was 5 µM. The y-intercept of Lines (A) and (B) is 0.9 sec/uM; the y-intercept of Line (C) is 0.3 sec/uM. The slope of Line (A) is 1.8 sec; the slope of Lines (B) and (C) is 0.6 sec. Calculate the Km of the reaction represented by Line (B).
Pyridoxal phosphate (PLP) is a coenzyme for the enzyme ornithine aminotransferase. The enzyme was purified from cells grow in PLP = deficient media as well as from cells grown in media that contained pyridoxal phosphate. The stability of the two different enzyme preparations was then measured by incubating the enzyme at 37°C for different lengths of time and then assaying for the amount of enzyme activity remaining. The following results were obtained. (a) Why does the amount of active enzyme decrease with the time of incubation? (b) Why does the amount of enzyme from the PLP deficient cells decline more rapidly?
Based on the Lineweaver-Burke plot attached. Kinetic data were generated in the (1) absence of any inhibitor, (2) presence of 15 µM of a reversible inhibitor, or (3) presence of 20 µM of a second (distinct) reversible inhibitor. Purified enzyme concentration was 5 µM. The y-intercept of Lines (A) and (B) is 0.9 sec/uM; the y-intercept of Line (C) is 0.3 sec/uM. The slope of Line (A) is 1.8 sec; the slope of Lines (B) and (C) is 0.6 sec. Calculate the Vmax of the reaction represented by Line (C). Show all mathematical work, please.

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