Need help, please. This question refers to experimentally determining a Michaelis-Menten curve for an enzyme.If all of the substrate appears to be converted to product for the rangeof substrate concentrations you have tested, which of the followingchanges would be most appropriate to try next, in order to generatemore informative data about Vmax and Km when repeating the assay?a.Vary the enzyme and the substrate concentrations.b. Incubate the reaction at a higher temperature.C. Shorten the reaction time.d. Use a different substrate.

Parameters such as Km and Vmax are used for comparing enzyme activities. If we know the initial rate…

Answered: This question refers to experimentally…

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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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?
What is the need of adding DNS solution to the enzyme substrate solution bWhat is the problem in determining rates at low substrate concentration? c. Calculate the Activity of an amylase enzyme which is diluted 1:100 times with phosphate buffer and incubated for 10 minutes at 37 degree Celsius. Given are the amount of maltose [mg] = 5.05 and the volume of enzyme used for the assay as 0.5ml.
what is the purpose of staggering the start and stop of the reactions? With reference to your experimental protocol, what is the purpose of staggering the start and stop of the reactions? A.To ensure that the reaction occurs with different amounts of enzyme in each tube so as to ensure comparability between reaction tubes. B.To ensure that the reaction occurs at exactly the same pH in each tube so as to ensure comparability between reaction tubes. C.To ensure that the reaction occurs for exactly the same time interval (30 minutes) in each tube so as to ensure comparability between reaction tubes. D.To ensure that the reaction occurs with exactly the same amount of substrate in each tube so as to ensure comparability between reaction tubes.
The initial velocity data shown in the table were obtained for an enzyme. Each assay at the indicated substrate concentration was initiated by adding enzyme to a final concentration of 0.01 nM. Derive Km, Vmax, kcat, and the specificity constant. [S] (mM) Velocity (x10^7) 0.10 0.96 0.125 1.12 0.167 1.35 0.250 1.66 0.50 2.22 1.0 2.63
reaction= Acetaldehyde +NADH +H+<-> ethanol +NAD+ Describe two procedural changes that would have to be made to this assay if you wanted to observe the reaction above. Explain why these changes would need to be made. Describe one analytical (data visualization and interpretation) change that would have to be made to this assay if you wanted to observe the reaction above. Explain why this change would need to be made.
During the early stages of an enzyme purification protocol, when cells have been lysed but cytosolic components have not been separated, the reaction velocity-versus-substrate concentration is sigmoidal. As you continue to purify the enzyme, the curve shifts to the right. Explain your results. This is an allosteric enzyme and you must use a Lineweaver-Burk plot to determine KM and Vmax correctly. 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 you purify away one of the substrates: This is an enzyme that displays Michaelis-Menten kinetics, and you must use a Lineweaver-Burk plot to determine KM and Vmax correctly.
One of your colleagues has obtained a sample of muscle phosphorylase b that is known to be relatively inactive. She has approached you for advice on how to set up an appropriate assay. She has the following items available, not all of which are appropriate for this study. Help her out by selecting the items that she should use and what their purpose is in the assay, and then explain why each of the other items would not be useful. 1. 100 UM AMP 2. 100 UM GTP 3.100 uM glucose 4. 100 uM glucose 6-phosphate 5. Branched glycogen 6. Amylose (i.e. unbranched glycogen) 7.50 mM HEPES buffer, pH 7.5 8. 50 mM potassium phosphate buffer, pH 7.5 Write out a short explanation for each of the above items, and upload your answers by the due date.
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?
Shown below are Km, and Vmax values obtained for an enzyme A which catalyze the transformation of the following substrates. Enzyme concentration used was 0.01 M. Km, mM 0.02 Vmax, mM/min 5.3 Substrate 1 2 1.5 13.7 3 2.6 100 4 0.1 25 0.05 62 1. Which substrate have the highest affinity for the enzyme? Explain. 2. Which will show higher efficiency of converting the substrate to the product? Show solutions and еxplain.
This assay is the most common method of detection in enzyme assays: A. рН B. spectrophotometric assay C. radiation D. electromagnetism The structure of THF comes from which derivatives: A. Pteridine derivative B. p-Aminobenzoic acid C. Glutamate D. All of the above Role of coenzyme, except for: A. Electron transfer reactions B. Allow precise orientation of the substrate at the active site C. Chemical group transfer reactions D. None of the above
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
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.

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