Case 4 - Enzyme Kinetics Case Spring 202 PDF

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This home work involves looking at the biochemical process of enzyme kinetics...

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Introduction to Biochemistry and Molecu Molecullar Biology BCMB 3100 57248

Ca se Study Case Spring 2021

Enzyme Kinetics Case

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Tak ake-H e-H e-Home ome P Point oint Not all enzymes work at the same rate and differences among enzymes help us understand the complexity of living things. Differences in the rates of enzymes can be described by determining, for example, how well they bind substrate and how fast they produce product. This field of biochemistry is called enzyme kinetics (i.e., enzyme rates). Enzymes can be inhibited. The kinetic properties of an enzyme in the presence or absence of an inhibitor can be determined to reveal how the inhibitor works.

Learning Objective Objectivess  Define Vmax, kcat and KM.  Identify Vmax and KM using numerical calculations of Michaelis-Menten equation.  Use Excel and grid paper to generate Michaelis-Menten and Lineweaver-Burk plots.  Identify Vmax and KM on Michaelis-Menten and Lineweaver-Burk plots.  Compare and contrast competitive, uncompetitive, and non-competitive inhibition.  Compare and contrast irreversible and reversible inhibitors.  Distinguish between different types of enzyme inhibitors using Lineweaver-Burk plots and calculations of V max and KM.  Predict the impacts that mutation would have upon an enzyme’s kinetic characteristics.  Understand the mechanism of Statins, a class of cholesterol lowering medications.

Pa Part rt 1 – Definitions 1. Start by defining the following terms: a. Vmax Insert you

Jin kyu Lee Fall 2019, Updated Spring 2021 by Jin Kyu Lee

b. KM Insert your answer

c. kcat Insert your answer

Pa Part rt 2 – Study of Enzyme-Catalyzed R Reaction eaction 2. To study the dependence of the rate of an enzyme-catalyzed reaction on the substrate, concentration, a constant amount of enzyme is added to a series of reaction mixtures containing different concentrations of substrate (usually expressed in moles per liter). The initial reaction rates are determined by measuring the number of micromoles of product produced per minute. Consider such an experiment in which the initial rates in Table 1 graphical) aphical) calculations in were obtained at the indicated substrate concentrations. Use numerical (not gr answering the following que quess tions. Table 1: Initial Rates at Various Substrate Concentrations of a Hypothetical Enzyme-Catalyzed Reaction [S] (mol/L)

Vo ( mol/min)

5.0 X 10

-2

0.25

5.0 X 10

-3

0.25

5.0 X 10-4

0.25

5.0 X 10

-5

0.20

5.0 X 10-6

0.071

5.0 X 10-7

0.0096

a. What is Vmax for this concentration of enzyme? Insert your answer b. Why is vo constant above substrate concentrations of 5.0 X 10-4 M? Insert your answer c. What is the concentration of free enzyme at 5.0 X 10-4 M substrate concentration? Insert your answer d. Calculate the KM of this enzyme using Michaelis-Menten equation. Show your work in detail.

Insert your answer e. Show that this reaction does or does not follow simple Michaelis-Menten kinetics.

Insert your answer f. What are the initial rates at [S] = 1.0 X 10-6 M and at [S]= 1.0 X 10-1 M? Insert your answer

Pa Part rt 3 – Enzyme Inhibition A biochemist studying the properties of a newly isolated metabolic enzyme obtains the following rate data during kinetic experiments in the absence and presence of three different inhibitors, A, B and C. A is an unknown chemical. B is a substrate analogue. C is an inhibitor that acts by binding to a lysine residue in the enzyme’s active site, to form a Schiff base, which is a covalent linkage.

[S] (nM)

Vo (mole/min)

Vo ( mole/min) With A present

Vo ( mole/min) With B present

Vo ( mole/min) With C present

50

0.14

0.055

0.077

0.067

75

0.19

0.063

0.11

0.085

150

0.32

0.086

0.19

0.14

400

0.47

0.10

0.36

0.24

3. Generate ONE graph (i.e., plot data for all experiments on the same graph), which depicts the initial velocities of the enzyme with and without inhibitors, as a function of substrate concentration. This is also known as a MichaelisMenten graph. You can do it on the grid paper or use Excel or another graphing program. Be sure to clearly label which data points represent no inhibitor, inhibitor A and B. A. Insert your Michaelis-Menten graph below. Insert your graph B. Estimate Vmax for the enzyme without and with inhibitors on your graph, and write those values here: Insert your answer

C. Estimate KM for the enzyme without and with inhibitors, and write those values here: Insert your answer

D. Why is it challenging to estimate Vmax and KM using a simple plot of initial velocities and [S]? Insert your answer

4. Generate ONE double-reciprocal plot (i.e., a Lineweaver-Burk plot). Be sure to clearly label which data points represent no inhibitor and inhibitors. Now you will need to use a graphing program. A. Insert your Lineweaver-Burk plot below. Insert your graph

B. Using this plot, determine Vmax values for the enzyme without and with inhibitors. Insert your answer

C. Using this plot, determine KM values for the enzyme without and with inhibitors. Insert your answer D. Do your responses to Q.3. B/C and Q.4. B/C differ? If so, explain why you think this is true. In other words, what is the advantage of a double-reciprocal plot over a Michaelis-Menten graph? Insert your answer E. Based on the information you have gathered and your answers to previous questions, determine the inhibition types for each inhibitor. Insert your answer

5. Suppose that there are individuals in the population that have a mutation in an enzyme gene. The residue at position 50, which is normally aspartate, is substituted with alanine (D50A). The D50A mutation occurs at an area within the active site that is important for substrate binding, but not for enzyme catalysis. a. Predict how this would affect the KM, Vmax, and kcat for the enzyme. Insert your answer b. Compared to what you learned about inhibitors is this case, what type of inhibition would this mutation resemble? Insert your answer 6. Cholesterol has a much higher profile than the other lipids because of its association with heart disease. Despite its lethal reputation with the public, cholesterol is vital to the body: it maintains proper fluidity of animal cell membranes and is the precursor of steroid hormones such as progesterone, testosterone, estradiol, and cortisol. Cholesterol is synthesized in the liver and, to a lesser extent, in other tissues. The rate of its synthesis is highly responsive to the cellular level of cholesterol. Cholesterol synthesis can be effectively blocked by a class of compounds called statins. The market of the statins is estimated at $20 billions. a. Explain the mechanism of action of statins. Insert your answer

b. Why has there been controversy about statins? Answer using the cholesterol synthesis pathway below.

https://commons.wikimedia.org/w/index.php?curid=21786963

Insert your answer

c. Should statins be used to lower cholesterol level? Insert your answer

Pa Part rt 4 – LUW Re Reflection flection (A) What are the two most important things you LEARNED from this case study? Insert your answer

(B) What are you still UNCLEAR about? Insert your answer

(C) What WORK will you do after class to clarify it? Insert your answer...