Chapter 4
ENERGY AND ENZYMES
All living beings are dependent on a continuous supply of energy in order to carry on
metabolism, maintenance, growth, and activity. Living cells utilize the potential energy stored in
the chemical bonds of organic molecules, in particular sugars and lipids, as their primary source
of energy. As the cell converts this chemical energy into usable forms, for instance to perform
cellular work, its supply of chemical energy must be continually replenished. This is
accomplished by the continuous movement of nutrients and energy rich molecules across the
cell membrane into the cell. Thus, the highly organized state of the cell is maintained by a
steady supply of nutrients which serve as a source of energy and raw material for the synthesis
of cellular components. All the metabolic reactions that define this organized state are
mediated by catalytic proteins called enzymes. Without the involvement of enzymes most
biochemical reactions would not occur at a measurable rate, and many substances might
engage in a variety of possible reactions instead of the one that is appropriate at a given place
and time.
I.
ENERGY CONVERSION IN BIOCHEMICAL REACTIONS
All chemical reactions are accompanied by a change in energy. If the energy of the
reactants (substrates) is higher than the energy of the products, energy is released during the
reaction. On the other hand, if the products have a higher energy than the reactants, energy
has to be supplied for the reaction to proceed.
In biochemical reactions, which occur under conditions of constant pressure and
temperature, the energy of reactants and products is expressed as the so-called free energy
(G). Whereas the absolute value of the free energy of a system cannot be effectively
measured, changes in free energy (∆G) that occur during a reaction can be determined. It is
very useful to a biologist to know this change in free energy of a given reaction, since ∆G is the
amount of energy that is available to do cellular work. Moreover, knowing the change of free
energy of a reaction allows a prediction about the reaction’s spontaneity. A negative ∆G means
that the reaction is exergonic and will occur spontaneously in the given direction. A positive ∆G
means that the reaction is endergonic and will not be spontaneous, but has to be driven with an
external source of energy (Figure 4-1). The source of energy that drives most endergonic
reactions in living cells is the bond energy of adenosine triphosphate (ATP).
Figure 4-1.
Changes of Free Energy in
Biochemical Reactions
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II.
ENZYMES
The reaction of most organic molecules with molecular oxygen is exergonic. For
instance, the oxidation of 1 mole of glucose (C6H12O6) with 6 moles of molecular oxygen (6 O2)
to 6 moles of carbon dioxide (6 CO2) and 6 moles of water (6 H2O) releases an energy of 686
kcal, i.e. ∆G = - 686 kcal/mol. This in enough energy to heat 2 gallons of milk from room
temperature to boiling temperature! Given the strong exergonic character of this oxidation, it
seems strange that there is any sugar existing in the presence of molecular oxygen. The
reason for the stability of sugar and other organic molecules in the presence of oxygen is the
so-called activation energy (or free energy of activation, EA), which has to be overcome by the
molecules of the reactants in order to react (Figure 4-2). At room temperature only a tiny
fraction of the molecules possesses a sufficient kinetic energy to overcome this “energy hill”,
therefore sugar and oxygen molecules do not react with each other under those conditions.
Without the presence of a catalyst most biochemical reactions are unmeasurably slow
because of their high activation energy. The living cell solves this problem by producing a
specific biocatalyst, an enzyme, for each reaction it has to carry out. Enzymes bind the
reactant molecules transiently in their active sites, bring them into close proximity of each other,
and arrange them in an orientation that favors their reaction. The result of this enzyme action is
an efficient reduction of the activation energy of a biochemical reaction (Figure 4-2). In some
cases the reaction speed is increased billion fold by the enzyme.
Two important features of enzymatic reactions should be kept in mind: 1) the enzyme is
not changed chemically in the reaction. After the reaction, the products dissociate from the
enzyme molecule, and the enzyme can bind new substrate molecules for a new round of
catalysis. 2) an enzyme can only speed up a reaction, it cannot make an energetically
unfavorable (endergonic) reaction happen. In case of an endergonic reaction, energy has to be
supplied to drive such a reaction “uphill” (Figure 4-1).
Figure 4-2.
Free Energy of Activation (EA)
without or with Enzyme
III.
LACTASE
The enzyme lactase, also called β-galactosidase, catalyzes the hydrolysis of βgalactosides. A naturally occurring substrate of lactase is lactose (milk sugar) which is found in
concentrations of up to 5 % in the milk of cows. Lactose is a disaccharide, made up of β-
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galactose and glucose (Figure 4-3). A deficiency in lactase activity causes lactose intolerance
in humans. After the consumption of milk products, lactose accumulates in the intestinal tracts
of affected individuals, due to the inability to hydrolyze the disaccharide and absorb its
components into the blood stream. Left undigested, lactose can lead to the production of gas,
bloating, diarrhea and stomach discomfort.
In this lab, we will measure the activity of a commercially available lactase (Lactaid),
which is used to help lactose intolerant individuals to digest lactose. We will compare the
activities of Lactaid Ultra (9000 units per caplet) and Regular Strength Lactaid (3000 units per
caplet), and we will determine the optimal pH and temperature for the enzymatic reaction, using
the synthetic substrate ortho-nitrophenyl-β-D-galactoside (ONPG).
ONPG is widely used to determine the activity of lactase. The enzyme hydrolyzes
ONPG, a β-galactoside, with a similar reaction speed as the natural substrate lactose.
However, after cleavage of ONPG the released molecules of ortho-nitrophenol (ONP) will
absorb light under alkaline conditions and turn the solution yellow (Figure 4-3). The intensity of
the yellow color is a direct measure for the concentration of ONP in the solution and, therefore,
for the amount of the substrate ONPG that has been hydrolyzed by the enzyme. The color
intensity is measured as so-called absorbance in a spectrophotometer (Spectronic 20) at a
wavelength of 420 nm.
Figure 4-3. Lactase-catalyzed Hydrolysis of Lactose and ONPG
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A.
Enzyme Concentration
The rate of an enzyme-catalyzed reaction depends on the concentration of the reactants
and the concentration of the enzyme. If the substrates are present in saturating amounts, there
is a linear relationship between enzyme concentration and reaction rate, i.e. doubling the
enzyme concentration will double the speed of the reaction. We will test this concentration
dependence of the reaction rate with two Lactaid products that contain different amounts of
lactase enzyme per caplet (9000 units vs. 3000 units).
PROCEDURE
WORK IN GROUPS OF FOUR THROUGHOUT THE ENTIRE LAB SESSION!
1.
Label two sets of 15 ml reaction tubes (3 tubes each) with the numbers 5, 10, or
15, respectively; label an additional tube with C (this will be your control).
2.
Add 2 ml of stop solution to each of the 7 tubes.
3.
Label two 25 ml Erlenmeyer flasks with E1 or E2, respectively; label another 15
ml tube with 0.
4.
Add 12 ml of the pH 4.5 reaction buffer (contains the substrate ONPG) to both
flasks; add 4ml of the reaction buffer to the tube labeled 0.
5.
Add 1 ml of enzyme solution 1 to the flask labeled E1 and 1 ml of enzyme
solution 2 to the flask labeled E2; mix by swirling.
6.
Incubate at room temperature.
7.
After 5 min, transfer 4 ml of the reaction mix from flask E1 to one of the tubes
labeled 5, and 4 ml of the mix from flask E2 to the other tube labeled 5; close the
tubes with the screw caps and mix by inverting; keep the tubes separated.
8.
After 10 AND 15 min, repeat the transfer of 4 ml of reaction mix from flasks E1
and E2; use the tubes labeled 10 and 15, respectively.
9.
Transfer the content of tube 0 to tube C; mix.
10.
Transfer the content of tube C to a glass cuvette; use this solution to set the
absorbance of the spectrophotometer at a wavelength of 420 nm to 0.
11.
Measure the absorbance of each of the other 6 solutions; use the same cuvette
for all of them.
12.
Record your data in the table below and plot them in the graph; fit a line through
zero and the first two data points, and calculate the reaction rate ∆A420/min.
13.
Rinse your tubes, flasks, and pipettes with tap water and use in next experiment.
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Table 1. ONPG Hydrolysis by Lactase Solutions 1 (E1) and 2 (E2)
t/min
A420
E1
E2
0
5
10
15
Figure 4-4. ONPG Hydrolysis by Lactase Solutions E1 and E2
QUESTIONS
1.
Which of the enzyme solutions contained more lactase activity?
2.
Is the ratio of the two reaction rates as you would expect from the manufacturers
information?
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B.
pH Dependence
The function of an enzyme is absolutely dependent on its three-dimensional
conformation. The substrates have to fit into the active sites of the protein, so that their
interaction is facilitated and their reaction can occur at a high rate. Any condition that alters the
shape of the enzyme will therefore interfere with the catalytic reaction.
The three-dimensional shape of an enzyme, its tertiary structure, is stabilized by weak
forces between amino acid side groups, such as hydrogen bonds and ionic interactions. These
interactions, in particular ionic bonds, are sensitive to pH changes in the environment of the
enzyme. Each enzyme exhibits a pH optimum, above and below which the reaction speed will
decrease. Under extreme pH conditions an enzyme will be irreversibly denatured.
PROCEDURE
1.
Label three sets of 15 ml reaction tubes (3 tubes each) with the numbers 5, 10,
or 15, respectively; label an additional tube with C (this will be your control).
2.
Add 2 ml of stop solution to each of the 10 tubes.
3.
Label three 25 ml Erlenmeyer flasks with 2.5, 4.5 or 7, respectively; label another
15 ml tube with 2.5.
4.
Add 12 ml of the pH 2.5 reaction buffer to the flask labeled 2.5, 12 ml of the pH
4.5 reaction buffer to the flask labeled 4.5, and 12 ml of the pH 7 reaction buffer
to the flask labeled 7; add 4ml of the pH 2.5 reaction buffer to the tube labeled
2.5.
5.
Add 1 ml of enzyme solution 1 to each of the 3 flasks; mix by swirling.
6.
Incubate at room temperature.
7.
After 5 min, transfer 4 ml of each reaction mix from the flasks to the tubes
labeled 5; close the tubes with the screw caps and mix by inverting; keep the
tubes separated.
8.
After 10 AND 15 min, repeat the transfer of 4 ml of reaction mix from each flask;
use the tubes labeled 10 and 15, respectively.
9.
Transfer the content of tube 2.5 to tube C; mix.
10.
Transfer the content of tube C to a glass cuvette; use this solution to set the
absorbance of the spectrophotometer at a wavelength of 420 nm to 0.
11.
Measure the absorbance of each of the other 9 solutions; use the same cuvette
for all of them.
12.
Record your data in the table below and plot them in the graph; fit a line through
zero and the first two data points, and calculate the reaction rate ∆A420/min.
Rinse your tubes, flasks, and pipettes with tap water.
13.
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Table 2. ONPG Hydrolysis by Lactase at Different pH Values
t/min
A420
pH 2.5
pH 4.5
pH 7
0
5
10
15
Figure 4-5. ONPG Hydrolysis by Lactase at Different pH Values
QUESTIONS
1.
At which pH value does Lactaid lactase work best?
2.
Which consequence does this pH optimum have for the enzyme’s therapeutic
use in lactose intolerance?
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C.
Temperature Dependence
The rate of any chemical reaction increases when the temperature increases. This
holds true in principle for enzymatic reactions too, but the stability of the enzyme limits the
extent of a beneficial temperature increase. If the temperature of the environment exceeds a
certain value, which is different for each enzyme, the weak interactions between the side chains
of the amino acids are broken and the three-dimensional shape of the protein collapses (heat
denaturation). This will obviously lead to a loss of catalytic function.
PROCEDURE
1.
Label three sets of 15 ml reaction tubes (3 tubes each) with the numbers 5, 10,
or 15, respectively; label an additional tube with C (this will be your control).
2.
Add 2 ml of stop solution to each of the 10 tubes.
3.
Label three 25 ml Erlenmeyer flasks with “ice”, 37 or 70, respectively; label
another 15 ml tube with 70.
4.
Add 12 ml of the pH 4.5 reaction buffer to each of the 3 flasks; add 4ml of the pH
4.5 reaction buffer to the tube labeled 70.
5.
Add 1 ml of enzyme solution 1 to each of the 3 flasks; mix by swirling.
6.
Incubate the flask labeled “ice” on ice, the flask labeled 37 at 37oC, and the flask
labeled 70 at 70oC; incubate the tube labeled 70 at 70oC.
7.
After 5 min, transfer 4 ml of each reaction mix from the flasks to the tubes
labeled 5; close the tubes with the screw caps and mix by inverting; keep the
tubes separated.
8.
After 10 AND 15 min, repeat the transfer of 4 ml of reaction mix from each flask;
use the tubes labeled 10 and 15, respectively.
9.
Transfer the content of tube 70 to tube C; mix.
10.
Transfer the content of tube C to a glass cuvette; use this solution to set the
absorbance of the spectrophotometer at a wavelength of 420 nm to 0.
11.
Measure the absorbance of each of the other 9 solutions; use the same cuvette
for all of them.
12.
Record your data in the table below and plot them in the graph; fit a line through
zero and the first two data points, and calculate the reaction rate ∆A420/min.
13.
Rinse your tubes, flasks, and pipettes with tap water.
4-8
Table 3. ONPG Hydrolysis by Lactase at Different Temperatures
t/min
A420
0oC
37oC
70oC
0
5
10
15
Figure 4-6. ONPG Hydrolysis by Lactase at Different Temperatures
QUESTIONS
1.
At which temperature does Lactaid lactase work best? Can you tell?
2.
With respect to its temperature optimum, is Lactaid lactase a good choice for the
treatment of lactose intolerance in humans?
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IV.
LAB REPORT
A significant part of scientific work is the publication of research data. You will therefore
write a lab report about part of this lab, following the format of a scientific paper. The lab
instructor will discuss with you the details of this format.
You should include in this report the dependence of the reaction rate on the
concentration of lactase (A) and one of the other measurements, pH dependence (B) or
temperature dependence (C).
Scientific reports follow a standardized format. They are generally composed of a
number of sections: Title and Author(s), Abstract, Introduction, Materials and Methods, Results,
Discussion, Acknowledgments, and References (Literature Cited). The exact requirements for
each of these sections may vary between different journals and publishers, and the authors
have to follow a journal-specific set of instructions to be considered for publication in that
particular journal (for example, have a look at the “Instructions to Authors” of the Journal of
Neurochemistry at http://www.jneurochem.org/misc/ifora.shtml).
The title of your report has to be descriptive and specific, it should be a one-sentence
summary of your work. The title of this chapter of the lab manual, “Energy and Enzymes,” does
certainly not fulfill these criteria of a good title for a research report. In the world of publications,
the title has to capture the interest of a potential reader, so that she/he will have a closer look at
your article.
The abstract, which immediately follows the title page, is the visiting-card of your report.
It must concisely summarize the essence of your report in a single paragraph and generally
with fewer than 200 words. It should contain the rationale of your work, the experimental
approach, the main results, and the major conclusions. The abstract should be self-contained
to an extent that others dare to cite your work without ever reading the full article.
In the introduction you give a brief summary of relevant background information. For
this lab, the introduction should include some information on enzyme catalysis and lactase
function. This background information will then lead to a statement of your objectives, i.e.
which specific problems are being addressed in the presented work. If appropriate, state the
hypotheses you set out to test at the end of the introduction section.
In your overview of relevant background information, you have to back up your
statements with citations of your sources. If you summarize information you found in your
textbook, for instance, you should cite the book at the end of the respective paragraph in the
format: (Campbell and Reece, 2002). Other sources to cite may be your lab manual or the
lectures.
In the materials and methods section you describe the materials you used for your
work and how you performed the experiments. This section is written in past tense. You do not
list what you were supposed to do according to your lab manual, but what you actually did! This
section has to be specific enough that anybody who wants to repeat the experiments would be
able to do so. Remember, scientific results have to be reproducible anytime and anywhere. Of
course, published procedures you used during your work have to be cited.
The results section is the “meat” of a scientific paper. Here you present your data in
the context of the performed experiments. A clear presentation of your results will include
tables and figures with descriptive captions (located at the top of a table, but at the bottom of a
figure). However, major results, such as the lactase-catalyzed hydrolysis rates of ONPG under
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varying conditions in this lab, must be summarized in the text of the results section. When
describing results in the text that are shown in or derived from a table or figure, always refer to
that table or figure. Avoid interpreting your data in this section of your report.
The results part of this lab report must contain the relevant tables and graphs found in
the lab manual. In addition, include figures that show the lactase activity (∆A420/min) as a
function of the tested variable. These figures may be bar diagrams showing lactase activity
under the conditions tested.
In the discussion section you interpret your findings in relation to your objectives and
your expectations based on available background information. This section of your paper
should also include an evaluation of possible sources of error. In the last paragraph of your
discussion you clearly state the conclusions of your study; this is the “take-home” message you
want EVERY reader to understand.
You may use the questions at the end of each experiment in this chapter as a guide for
your discussion. However, do not limit yourself to the issues raised by those questions. For
instance, if you discover a flaw in the design of a particular experiment, point it out and suggest
an improved experiment.
In the acknowledgments you thank persons who contributed to the study, but do not
occur as coauthors on the paper. In our case, the acknowledgment section should include
Mark Damie, the lab technician who makes sure that this BioI lab is equipped with all the
necessary machines and supplies to perform the experiments. You may also want to mention
your lab instructor, if you discuss details of your report with her/him before submitting it, or
student peers who were helpful with discussions that enabled you to write a better paper.
At the end of a scientific report you will find a references section, in which publications
that were cited in the text are listed, frequently in alphabetical order. For this lab report, please
cite in alphabetical order following the format below:
Book: Campbell, N. A. and J. B. Reece. 2002. Biology, 6th ed. Pearson Education, CA,
pp. 96-100.
Lab Manual: Krieger, K. and U. Pott. 2002. Bio I Laboratory Manual. Energy and
Enzymes, pp. 4-1-4-9. UWGB, WI.
Journal Article: Colello, R. J. and U. Pott. 1997. Signals that initiate myelination in the
developing mammalian nervous system. Mol. Neurobiol. 15: 83-100.
Web Pages: National Institute of Standards and Technology, Physics Laboratory, year.
“International Systems of Units (SI).” http://physics.nist.gov/cuu/Units/index.html (October 1,
2000).
A word of caution about citing web pages: you should only cite web pages from reliable
sources, such as electronic journals or web sites maintained by recognized scientific
authorities. Since the structure and content of web sites may change frequently, you must
indicate the date of access to the web site and the date the information was posted (if known; in
the above example, the date of posting is unclear). If the author of a web page is known, you
should cite her/his name rather than the institutional name.
Personal communication: The information you gather on a specific subject in lecture has
the quality of a personal communication, unless the lecture notes are published in some way.
Personal communications can be cited in the text, usually with consent of the person who gave
the information, but are not listed in the references section.
The above order of sections is not the order in which you should write your report. You
will generally begin with the parts you know best, i.e. the materials and methods, and your
results. Writing these chapters will put you in the right mind-set for tackling the more complex
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tasks of writing the discussion and the introduction. Since the abstract is a concise summary of
the whole paper, it should definitely be written last.
For a more careful discussion about writing a research article consult “A Short Guide to
Writing about Biology” by Jan A. Pechenik (see recommendation in syllabus).
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