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

| October 3, 2018

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:.youtube.com/watch?v=YAva4g3Pk6k”>here (video 2min) and.youtube.com/watch?v=y-uuk4Pr2i8″>here (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.

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