Enzyme Kinetics – The Pre-Lab assignment is all that needs done

26 Jun Enzyme Kinetics – The Pre-Lab assignment is all that needs done

Question
Pre-Lab (2 pt each)

1. Consider the reaction: Glucose + hexokinaseà glucose-6-phosphate

Assign the molecule as the enzyme, substrate, and product

Glucose = __________________

Hexokinase = _______________

Glucose-6-phosphate= ________________

2. Which of the above reaction components corresponds to A, B, and E in the online tutorial?

Glucose = _________________

Hexokinase = ______________

Glucose-6-phosphate= ____ ___________

3. Fill in the blank:

When the reaction between glucose and hexokinase reaches equilibrium, the reaction rate, v, is ________________because the reaction is moving ______________________.

4. How would you describe the relative proportions of substrate and enzyme in a biological reaction?

5. What is the “ceiling” that limits the rate at which an enzyme can convert a substrate into a product?

_______________________ ____

6. What does the Michaelis-Menton (M-M) curve describe? Why do scientists care about the M-M curve?

7. What does the flattening of the Michaelis-Menton curve signify with the respect to the enzyme active site and substrate concentration?

8. Referring to Section 5 of the tutorial:

If the Km and Vmaxof Enzyme 1 are 16uM and 120uM/min, respectively, and Enzyme 2 has Km=11.8 microM, which enzyme has a stronger affinity for a given substrate?

If Vmax stays the same and Km decreases, does the reaction rate reach Vmax faster or slower?

9. What does “hyperbolic” mean in regards to the Michaelis-Menton plot?

10. True or False? When a reaction reaches equilibrium, product is no longer formed.

11. True or False? The Michaelis-Menton plot is a graphic representation of the product formed over time.

12. If you answered FALSE to questions 10 or 11, explain why.

Question 10. _____________________________________________________

Question 11. _____________________________________________________

13. Lysozyme is an enzyme that breaks down bacterial cell walls. Its Km is 6 mM. Chymotrypsin is a digestive enzyme that breaks peptide bonds and its Km is 5000 mM.

a) Insert the Km values for the two enzymes in the M-M curves below. Use a line to indicate where Km crosses the y- or x-axis.

b) Which of these two enzymes has the higher affinity for its substrate and why do you think so?

c) Which enzyme(s) will be operating at maximum efficiency at the following concentrations of substrate?

Write L, C, B, or N in the box to the right of the substrate concentration, where lysozyme only (L), chymotrypsin only (C), both (B) or neither (N).

1 mM

100 mM

50,000 mM

6 mM

5000 mM

Questions on article:

14. Make a schematic which includes the general shape of the motor protein kinesin, including the cellular components that bind to kinesin. (Will need to look up the function of kinesin in your text book.) Hint: Kinesin is a polar molecule, meaning that each end has a specific function. What are those functions?

15. Why is kinesin considered an enzyme?

16. How does mutant kinesin promote muscle weakness?

17. NOW complete the Experimental Design sections in Lab Activities (6pt)

Specific Information for this lab

In this lab you will first practice recording enzyme kinetic data using the enzymeb-galactosidase as a model enzyme. You will then apply this knowledge to study the enzyme kinetics of kinesin, a cellular motor protein.b-galactosidase offers analytical and visual information to study the conversion of a substrate to a product, because the substrate, ONPG, is clear, but the product , o-nitrophenol, is yellow. b-galactosidase is a bacterial enzyme which has been studied extensively for decades. The basic reaction catalyzed by this enzyme in bacteria is:

You will be using a colorimetric assayto measureb-galactosidase activity.

Watch the video “.youtube.com/watch?v=pxC6F7bK8CU”>How does a spectrophotometer work?” to see how the “spec” instrument is used detect color change in a colorimetric assay.

In this assay, a colorless substrate (ONPG, or o-nitrophenyl-b-D-galactopyranoside) is converted to a colored product (o-nitrophenol),which absorbs light at wavelength 420 nm and can be measured using a spectrophotometer. The molar absorptivity of o-nitrophenol =0.0045 OD/nmol. You can use this information to calculate the amount of product formed in your reaction.

Activity 1: Investing the Activity of ?-galactosidase

By monitoring the appearance of the product o-nitrophenol (yellow color) over time you will quantitatively examine the rate at whichb-galactosidase catalyzes the conversion of ONPG to o-nitrophenol.

****Read through the procedures and create outlines for your data tables and/or figures before coming to lab. Your group will compile table/figures before beginning the procedure to make sure everyone is on the same page****

1. Construct the framework of a data table that will allow you to record the activity of ?-galactosidase every minute over a 10 minute time period. To do this, consider what data you will collect and what your column headings should be.

2. Add experimental data to the table you generated above using the reaction between 3 ml of 0.2 mM ONPG mixed with 100uL enzyme.

3. Plot your results on the axes below. Add appropriate units to each axis.

4. Label the part of the above curve where the rate of the reaction is highest.

5. Label the part of the above curve where the rate of the reaction is lowest.

6. The highest rate is ___________________________ (estimate value, include units)

7. The lowest rate is ____________________________(estimate value, include units)

8. Now, convert your raw OD versus time data into rate (OD/min) versus time data, by calculating the rate of the reaction for each 1 minute interval:

Enter Table of data for rate of o-nitrophenol production at 0.2mM ONPG here:

9. Plot the rate of the reaction as a function of time, using the axes below. Include units on each axis:

10. What do you conclude from this experiment? Do all time intervals provide equally reliable measurements of the reaction rate between beta-galactosidase and 0.2 mM ONPG? Why or why not?

Activity 2: Effect of Substrate Concentration on the Reaction Rate

For this section, you will measure the activity of ?-galactosidase at several different substrate concentrations and construct Michaelis-Menton curve from your data. This is the only way to ESTIMATE the Km and Vmax of the enzymeb-galactosidase for the substrate ONPG.

1. Write the protocol that you will follow to measure the rates of the reactions set up in the table below. Measure enzyme activity over a 20 minute period, and decide what time intervals will you use to determine the rate of the reaction in each tube. Also construct a data table to record your results.

Enter protocol here:

Enter Data table here:

2. Set up fourb-galactosidase/ONPG reactions as follows (DO NOT ADD enzyme until you are ready to put the reaction into the spec. When ready, add 100uL enzyme to tube, invert to mix, pour in cuvette, and place in spec):

Rxn 1

Rxn 2

Rxn 3

Rxn 4

2 mM ONPG

3.0ml

1.5ml

—

—

0.2 mM ONPG

—

—

3.0mL

1.5mL

Buffer

—

1.5mL

—

1.5mL

3. What is the initial substrate concentration in each of the reaction mixtures?

Rxn 1

Rxn 2

Rxn 3

Rxn 4

Initial [ONPG] (units?)

4. Before you actually do the experiment, predict what will happen in the reactions from step 2 by plotting points that you expect on the axes below. Add the appropriate units to the axes.

5. Run the reactions according to your protocol. Plot results on the axes provided. Add the appropriate units to the axes.

6. If you think that your data are unreliable, alter your protocol and make the measurements again.

7. Label the parts of your curve where the reaction rates are highest and lowest.

8. Is the reaction rate equally sensitive to all changes in substrate concentration? Why or why not?

9. Where the rate is highest, estimate the fraction of enzyme molecules that are in complexes with substrate?

10. Does this rate approximate Vmax for beta-galactosidase? Why or why not?

Activity 3: Mutations in the enzyme kinesin lead to loss of motor control and neuropathy

NOTE: This portion of the lab is performed virtually on computer.

Scenario

You are an undergraduate researcher in a neuroscience lab that studies disorders in muscle motor control. Tamara, a postdoc in your lab, studies inherited genetic mutations in kinesin, a motor protein that “walks” along microtubules to deliver proteins and vesicle cargo down the length of neurons (Essential Cell, 577-578). To introduce you to the kinesin protein, Tamara tells you to check out these videos:here (video 2min) andhere (video: 22 sec). Tamara is excited about a recent study she read that characterizes different mutants of kinesin and discusses the role of kinesin mutants in hereditary spastic paraplegias (HSPs), a group of neurodegenerative disorders characterized by spastic weakness of the lower extremities. She has acquired a sample of mutant kinesin from the authors of this paper and is performing a number of biochemical experiments on it.

Your project

Your project in the lab is to determine the enzyme kinetics, Vmax and Km, of normal and mutant kinesin using ATP as the substrate. You are excited because if you succeed you will be included as an author on Tamara’s manuscript. This would be a great resume booster and you want the lab to hire you as a summer intern, so you are trying to do your very best.

Tamara’s exciting reference

Peter Fuger, et al. Spastic Paraplegia Mutation N256S in the Neuronal Microtubule Motor KIF5A Disrupts Axonal Transport in a Drosophila HSP Model. PLoS Genetics, 2012. 8(11): 1-20.

Find on Bb under Lab Documents.

Procedure

Access the.wiley.com/college/pratt/0471393878/student/animations/enzyme_kinetics/index.html”>“Virtual Kinetics Experiment” in the Enzyme Kinetics tutorial and follow the prompts to perform the kinesin kinetics experiment virtually.