Download ENZYMES: PROPERTIES OF B

ENZYMES: PROPERTIES OF CHOLINESTERASES
W. Straus, J. Mayne, and L. Pezzementi
"Enzymes are protein molecules, a different kind to catalyze each
chemical event in every metabolic pathway by which cells
manufacture energy, structures, wastes, other enzymes, more DNA
and new cells."
From: H.F. Judson. The Eighth Day of Creation.
Simon and Schuster, N.Y. 1979
OUTLINE OF EXPERIMENT
Weeks 1-2. Molecular and Protein Modeling Using Spartan ’04 and MOE
Week 3.
Measuring Rate of Reaction, Effect of Substrate Concentration on
Rate of Hydrolysis I: Hydrolysis of Acetylthiocholine, II: Hydrolysis
of Butyrylthiocholine,
Week 4.
Data Analysis and Experimental Design for Cholinesterase Present
in Horse Serum
Week 5.
Student Experiments
INTRODUCTION
In this laboratory, we will be investigating some of the properties of the protein
molecules called enzymes, which function in cells as biological catalysts. A
catalyst is a substance that lowers the energy of activation for a reaction, without
being chemically altered. Although the specific case of cholinesterase is
illustrated in Figure 3, there are many different types of catalysts and there are
many different mechanisms by which catalysts lower the energy of activation for
a chemical reaction. So, before we begin examining cholinesterases, we will first
review the general principles behind enzymes, the most common type of catalyst
found in biological systems.
ENZYMES
As stated above, enzymes are proteins that act as catalysts. The temperatures
found at the surface of the earth are ideal for maintaining the structure of
proteins, membranes, and other macromolecular assemblies. However, without
enzyme catalysts, most of the chemical reactions that occur in cells would
proceed so slowly at these temperatures that they would take hundreds or
thousands of years to reach equilibrium. Thus, enzymes make it possible for
metabolic reactions to occur at temperatures where the structural components of
cells are reasonably stable.
48
Enzymes are usually very specific with respect to the reactants that they interact
with, they are active in small amounts, and they do not alter the equilibrium of the
system. There are thousands of different enzymes found in any given cell and
they catalyze thousands of different reactions by many different mechanisms. In
a typical reaction, the starting reactant(s) or substrate(s) undergoes a chemical
change and is converted into a product. Each enzyme has a unique chemical
structure and shape which allows it to catalyze a very specific chemical reaction.
Most, but not all enzymes are named after their substrate(s), with the suffix "ase"
at the end of the name. The names of enzymes also usually include some
reference to the type of reaction (e.g. esterase, isomerase, etc.). Some
exceptions to this rule include the digestive enzymes trypsin, chymotrypsin,
acrosin, thrombin, and pepsin.
Each enzyme shows specificity because its shape, structure, and charge is
such that it will only interact with a specific substrate or with molecules that
closely resemble the substrate. The enzyme combines with the substrate to form
an enzyme-substrate complex. The substrate is then converted to product,
leaving an enzyme-product complex, the product dissociates from the enzyme,
and the enzyme is ready to interact with another molecule of substrate:
E + S <===> ES <===> EP <===> E + P
Keep in mind that all enzymatic reactions are reversible, so if the concentration of
the product is high and that of the substrate is low, the reverse reaction can
occur. If the forward reaction is exothermic, the probability of the reverse reaction
occurring spontaneously is very low, since addition of energy to the system is
necessary for the product to be converted back into substrate. Conversely, if the
forward reaction is endothermic, the probability of the reverse reaction occurring
increases as the concentration of product goes up (refer to your lecture notes
and your textbook’s discussion of equilibrium). You must also remember that
some reactions behave as if they are irreversible.
The property that most enzymes have in common is that they are polymers of
amino acids that act something like springs. Each enzyme has a substrate
binding site, called the active site, located within folds at the surface of the
protein. When a molecule of substrate binds to the active site of an enzyme, it
distorts the shape of the enzyme. The distorted enzyme in turn places stress on
the chemical bonds of the substrate, thereby increasing the probability of the
bonds breaking and reforming. If two reactants are involved, they are brought
together in space when they bind to the active site. So, the enzyme performs the
same function as a high energy collision: it brings the reactants together in space
at just the right angle and places stress on the chemical bonds. Consequently, an
enzyme increases the probability of a chemical reaction and eliminates the need
to add energy in the form of heat to achieve activation. Enzymes usually bind to
substrates through weak interactions such as ionic bonding, hydrogen bonding,
Van der Waals forces, and hydrophobic interactions. Some enzymes actually
form covalent bonds with substrates during intermediate stages of a reaction.
Many factors influence the rate of product formation in an enzyme-catalyzed
reaction. For example, when the concentration of substrate is increased, the rate
of the reaction increases. There is a point when increasing the concentration of
substrate will saturate all of the available enzyme active sites and the rate of
reaction will no longer increase. This point is called the maximal velocity, or
49
Vmax. At Vmax, the rate of the reaction can only be increased by adding more
enzyme. Vmax represents the maximum amount of substrate that can react in
one minute (moles of substrate/min) and is a measure of the ability of an
enzyme to catalyze a reaction (catalytic efficiency).
Factors that influence the structure of an enzyme will often affect the ability of an
enzyme to catalyze a reaction. Increasing temperature increases the rate of all
chemical reactions. Beyond a certain temperature, however, enzymes lose their
ability to catalyze reactions because heat induces changes in their tertiary or
quaternary structures. Other factors that can alter enzyme structures include the
pH (hydrogen ion concentration) of a solution, ionic strength of a solution (the
concentration of dissolved ions), and divalent cations such as calcium or
magnesium.
Often, enzymes will bind specific chemicals that either inhibit or stimulate the rate
of the enzyme-catalyzed reaction. Inhibitors that reversibly bind to the active site
of the enzyme and thereby prevent the binding of the substrate, are called
competitive inhibitors because they compete with the substrate for the active
site. Inhibitors that bind to other parts of an enzyme and cause inhibition by
altering the shape of the enzyme are called allosteric or non-competitive
inhibitors. Many enzyme inhibitors occur naturally in cells and play important
roles in regulating various chemical reactions.
Biochemists use two important parameters when studying enzymes: the ability of
the substrate to bind to the active site (sometimes called enzyme-substrate
affinity), and the catalytic efficiency (Vmax). Factors such as temperature, pH,
ionic strength, and inhibitors can alter these parameters and give important clues
concerning the properties and regulation of a particular enzyme.
ENZYME ACTION: CHOLINESTERASES
All jawed vertebrates possess two evolutionarily related cholinesterases (ChEs)
as the result of a gene duplication event early in vertebrate evolution. One of
these ChEs is acetylcholinesterase (AChE), which hydrolyzes the
neurotransmitter acetylcholine (ACh) into acetate and choline at the
neuromuscular junction and at many synapses in the central and peripheral
nervous system, and terminates the action of the neurotransmitter; at the
neuromuscular junction, this hydrolysis allows the relaxation of skeletal muscle.
The other ChE is butyrylcholinesterase (BuChE). BuChE is widely distributed in
many tissues, with particularly high levels present in the liver. Despite the fact
that BuChE has been studied for well over 50 years, its function remains
uncertain. Certain humans appear to lack the enzyme, yet appear to be normal.
However, it is now widely thought to be involved in detoxification systems in
animals, hydrolyzing natural toxins that may be ingested. The two ChEs may be
distinguished by kinetic (substrate specificity and inhibition) and pharmacological
(diagnostic inhibition) analyses (Table 1), as well as by comparison of DNA and
amino acid sequences (Massoulié et al., 1993; Chatonnet and Lockridge, 1989).
In the past, BuChE was sometimes referred to as pseudocholinesterase, or just
cholinesterase. Today, butyrylcholinesterase is the official name,
pseudocholinesterase is no longer used, and cholinesterase refers to
cholinesterases in general.
Table 1. Characteristics of Vertebrate Cholinesterases.
50
Characteristic
Substrate Specificity
Acetylcholine
Butyrylcholine
Vetebrate AChE
Vertebrate BuChE
Yes
No
Yes
Yes
Substrate Inhibition
Yes
No
[Low]
[High]
[Low]
[Low]
[Low]
[High]
Diagnostic Inhibition
Eserine
Example inhibitor X
Example inhibitor Y
Substrate Specificity. As outlined in Table 1, AChE hydrolyzes ACh almost
exlcusively, while BuChE is capable of hydrolyzing both ACh and butyrylcholine
(BCh) at least as well. Thus, substrate specificity can be used to distinguish
between the two enzymes. Most commonly this comparison is made by
calculating Vmax ratios.
For AChE VmaxBCh/VmaxACh ≈ 0.01. For BuChE VmaxBCh/VmaxACh ≈ 1.0 to 2.0.
One should note that while ACh is a natural substrate present in vertebrates,
BCh is a synthetic compound - it does not exist in nature and is not a
neurotransmitter, but is used in enzymatic studies. BCh is a larger choline ester
than ACh as a result of its three-carbon, butryryl acyl group in place of the onecarbon, acetyl acyl group of ACh. The structures of ACh and BCh are shown in
Fig. 1. Note that the two structures are identical except for the acyl groups
located at the right side of the molecules (-CH3 for ACh and –CH2CH2CH3 for
BCh). In fact, this and other results indicate that BuChE has a larger active site
than AChE and is able to accommodate a relatively wide variety of substrates; it
is not nearly as specific as AChE. For example, BuChE can hydrolyze aspirin,
cocaine, and heroin.
Figure 1. Structures of acetylcholine and butyrylcholine. Acetylcholine is at the top and
butyrylcholine at the bottom. Since the two cholines contain positively charged quatenary
ammonium ions, they must have a negative anion.
51
Substrate Inhibition. BuChE follows Michaelis-Menten kinetics. AChE does not
follow classical Michaelis-Menten kinetics, but shows an additional kinetic
feature, inhibition of catalysis at high substrate concentrations, or substrate
inhibition (Table 1). AChE is an allosteric enzyme: at low substrate
concentrations, substrate binds only to the active site and is catalyzed to product;
at high substrate concentrations, substrate also binds to a second or allosteric
site, and this binding causes a conformational change in the protein, which
inhibits enzyme catalysis. BuChE does not exhibit substrate inhibition, and this
difference can be used to distinguish the two enzymes.
Diagnostic Inhibition. All ChEs are inhibited by the drug eserine (also called
physostigmine) at very low concentrations (μM). In contrast, other
pharmacological agents are more or less specific for either AChE or BuChE, and
differences in inhibition can be used to distinguish AChE from BuChE (Table 1).
52
CATALYSIS BY
CHOLINESTERASES
In the enzymatic mechanism
shown to the right you can see that
three amino acids (also known as
the catalytic triad) are involved in
catalysis by cholinesterases. In the
case of AChE, these amino acids
are the glutamate at positin 327,
the histidine at position 440 and the
serine at position 200.
First, a covalent bond is formed
between the acetyl group of the
substrate and serine 200.
Next, the bond between acetate
and choline is broken and choline
is released.
Finally, the covalent bond between
serine 200 and the acetate portion
of the substrate is broken, forming
acetate which is released from the
enzyme, and returning serine to it’s
original state so that it is available
for a new round of catalysis.
Throughout this diagram, the
movement of electrons is depicted,
as is the formation and breakage of
other critical bonds. Through this
movement of electrons glu327
activates his440, which in turn
activates ser200 so it can attack
the acetyl group of the substrate.
In the upcoming molecular
modeling exercise, you will locate
the active site of cholinesterase
and see exactly where in the
enzyme this reaction takes place.
Figure 3. Catalytic mechanism of ChEs
in the hydrolysis of choline esters.
Shown is the hydrolysis of ACh by a ChE.
53
Weeks 1-2: Molecular and Protein Modeling
using Spartan ’04 and MOE
Introduction to Molecular Modeling
Molecular modeling is a term that refers to the process of using mathematical calculations to
simulate various chemical events at the molecular (and sometimes subatomic) level. By
constructing models, scientists are better able to visualize how a molecule’s structure
influences its function, and thus they can design meaningful hypotheses to take into the lab
for further testing. Whereas some modeling approaches generate chemical structures from
scratch (ab initio) by relying solely on mathematical formulas that predict the probable
locations of electrons, other approaches synthesize experimental data to develop a working
model. Some common types of information used to build such a model include experimental
determinations of electron densities, atom sizes, and bond lengths. These data can be
generated by a variety of methods including x-ray crystallography, a technique described in
your textbook.
Due to the size and complexity of most biologically important molecules and
macromolecules, biologists that use molecular modeling in their research rarely use ab initio
methods; instead they often employ programs that rely on molecular mechanics to simulate
chemical structures and interactions. In molecular mechanics, atoms are represented as
hard spheres, and bonds are treated as being analogous to springs. Thus many of the laws
of classical mechanics (such as those developed by Newton) can be applied. Additional
factors that are taken into account computationally include the relative charge of the atoms in
a molecule and the type of environment in which a molecule is found (gaseous, aqueous, a
lipid bilayer, etc.).
An important feature of molecular modeling programs is the ability to change aspects of the
molecule being studied. For example, it is possible to add, remove, and alter specific atoms
and subsequently predict the effect of this change on a molecule’s structure. When
modeling enzymes, the user can alter specific amino acids in a given protein and the
resulting model can be used to predict which changes are likely to impair substrate binding
and catalysis. Alternatively, researchers can model inhibitors or other drugs and predict
which will bind most efficiently to a specific enzyme target.
Another critical feature of molecular modeling is the incorporation of stochastic elements.
The term “stochastic” refers to the fact that there is some randomness in a given calculation.
Although much is known about the factors that influence protein folding, it is not yet possible
to predict a protein’s structure with complete confidence, based solely on its primary
sequence of amino acids. Thus when considering a specific bond or the angle between
three specific atoms, molecular modeling programs must often make a choice, essentially
“flipping a coin” to choose between two or more possible values for a given parameter. This
computational “decision-making” results in variability in the outcomes generated. Thus, like
all good experiments, molecular modeling experiments should be repeated so that the
standard deviation in a model can be calculated.
Despite the intense and challenging mathematics behind molecular modeling, scientists at all
levels of training can use this technique thanks to the many user-friendly software platforms
have been developed, including HyperChem, CAChe, Alchemy, and Accelrys. As part of
your research on the cholinesterase in horse serum, you will use the molecular modeling
programs Spartan, CASTp, and Molecular Operating Environment (MOE). These
applications will help you to visualize the structure of cholinesterases, while examining the
amino acid residues important in catalysis and inhibitor binding.
54
Part 1: Building a Protein
For this stage of the experiment you will use the Molecular Operating Environment
(MOE) to create a simple protein. The example used is the hormone oxytocin, which
plays a key role in milk production and smooth muscle contraction during labor. The
protein is nine amino acids long. This protein serves only as a simple and short example
to illustrate the basic building and manipulating functions of MOE.
A) Open MOE by double clicking the
MOE icon on the desktop. The main
MOE screen will open.
B) To begin creating a protein click on
the “Edit” menu on the top toolbar.
From the drop down menu select
“Build” and then “Protein.” This will
open the protein builder (Shown at
right).
C) Using the buttons in the Residue
half of the protein builder, enter the
following sequence of amino acids by clicking their corresponding button: CYS, TYR, ILE,
GLN, ASN, CYS, PRO, LEU, GLY. Make sure that the amino acids are selected in the
right order. This will create the initial polypeptide chain in the MOE window.
D) Press “View” in the Protein Builder to center the chain, and then close the Protein Builder
by selecting “Close.”
E) View the structure of the chain. The protein can be rotated by holding the center mouse
button and dragging the mouse. The zoom is controlled by the center mouse wheel.
F) Now you will select two sulfur atoms by clicking “Selection” from the top toolbar. Choose
“Atom Selector” from the drop down menu. The Atom Selector will appear.
G) Click on “Table” under the Element section of the Atom Selector. Next select S from the
periodic table. Close the table and the Atom selector. Atoms appear pink when selected.
H) Select “Label” from the right button bar in MOE window. Choose “Element” from the drop
down menu. Notice the two sulfur atoms in the protein are labeled with a “S.”
I) Click on “Edit” from the top toolbar.
Select “Build” then “Molecule” from the
drop down menus. This will open the
Molecule Builder (Shown right).
J) Select the Single Bond button (Circled
in picture to right). You have now
created a disulfide bond. It should
appear to stretch nearly the length of
the entire chain. Close the Molecule
Builder.
K) Double click on any atom in the protein.
The Atom Manager will appear on the
screen.
L) Select “Compress All” in the Atom
Manager to reduce the amount of
visible information.
M) Double Click on “Chain 1.”
N) Double Click on “Residue 9 GLY”
O) From the list of named atoms, select the “OXT” atom.
P) Turn on the “Selected” option at the bottom of the manager, then click “Apply.”
Q) Turn on the “Selection Only” toggle button at the top of the Atom Manager.
R) We are only concerned with the atom OXT in Residue 9. Deselect any additional atoms
by first double clicking the residue line, and then double clicking the unwanted atom.
S) Close the Atom Manager
55
T) Re-open the Molecule Builder by selecting “Edit”- “Build”-“Molecule”
U) Choose N from the Molecule Builder to replace the OXT atom with a nitrogen atom.
Close the Molecule Builder. The replacement of the terminal oxygen with a nitrogen atom
occurs in oxytocin, but is not seen in most proteins.
V) Select “Compute” menu from the top toolbar in the MOE. Select “Partial Charges” from
the dropdown menu. Press OK. No visible changes should occur.
W) Select “Compute” again from the top toolbar. Select “Energy Minimize” from the
dropdown menu. Press OK (without changing any of the default settings). The protein
should fold into its most stable conformation.
Question 1: Describe the structure of oxytocin before and after minimization. Why might this
change occur?
Question 2: What color is associated with carbon in MOE? Nitrogen? Oxygen? Sulfur? This
color scheme is constant for every molecule observed in MOE.
X) When finished viewing the folded protein, close by selecting the “Close” button from the
right button bar. This will close the open screen without closing MOE.
Part 2: Acetylcholinesterase
MOE has many tools for examining and analyzing protein structures. This stage of the
lab will use MOE to explore the overall and secondary structures, as well as the
hydrophilic and hydrophobic nature of the enzyme acetylcholinesterase. It will also be
used to isolate and view the catalytic triad and the active site of the enzyme.
1) View the overall structure of AChE.
A) Open the acetylcholinesterase file by going to the “File” menu on the top toolbar and
clicking “Open.” Double click on the “Moe files” directory in the main screen. Then
open the “AChE.moe” file by double clicking it.
B) To see the enzyme, click the “View” button from the right button bar. This will center
and scale the enzyme to fit the window.
C) View the structure of the enzyme. The enzyme can be rotated by holding the center
mouse button and dragging the mouse. The zoom is controlled by the center mouse
wheel.
2) View the secondary structure of AChE.
A) Click “View” on the right button bar to center AChE.
B) Open the Sequence Editor by pressing the Ctrl and Q keys simultaneously.
C) Open the “Display” menu from the top toolbar of the Sequence Editor. Select the
“Actual Secondary Structure” toggle. Amino acid sequences that are part of an alpha
helix have a red line above them while amino acid sequences that are part of a beta
sheet have a yellow line above them. You may disregard any other colors that
appear.
Question 3: Describe the arrangement of alpha helices and beta sheets within the linear
sequence of amino acids making up acetylcholinesterase. How many stretches of
amino acid sequences are involved in alpha helices? How many stretches of amino
acid sequences are involved on beta sheets? Are all the amino acids involved in
beta sheets adjacent to each other? Are the stretches of alpha helices and beta
sheets alternating?
D) Close the Sequence Editor.
E) Open the “Render” menu from the top toolbar. Go to the “Backbone” submenu, and
click on the “Flat ribbon” option to show the secondary structure of the enzyme.
F) To hide the stick structure of the enzyme, click the “Hide” button on the right button
bar. Select the “All” option.
G) The view should now only show the secondary structure (Alpha helices and beta
sheets).
Question 4: Describe the arrangement of alpha helices and beta sheets in the threedimensional structure of acetylcholinesterase.
56
H) Replace all of the stick structure of AChE by clicking on the “Show” button on the right
button bar, and selecting “All.”
I) Remove the secondary structure by opening the “Render” menu from the top toolbar,
going to the “Backbone” submenu, and selecting “None.”
3) View the hydrophobic/hydrophilic residues of AChE
A) Begin by opening the Sequence Editor. Do this by pressing the Ctrl and Q keys
simultaneously. This editor shows all of the amino acid residues in the order that they
appear in the polypeptide chain.
B) Open the Residue Selector by opening the “Selection” menu from the top toolbar of
the Sequence Editor and selecting “Residue Selector.”
C) Once the Residue Selector is open, begin by clicking “Clear.” This will clear any
possible data left on the Selector.
D) Click on the “Hydrophobic” button at the bottom of the Selector. Then click “Select
atoms.” This will select all of the atoms in the enzyme that are the part of
hydrophobic residues.
E) Highlight the hydrophobic atoms by clicking the “Render” menu from the top toolbar
and selecting “Space-filling.” Then click the “Render” menu, then “Color,” and then
select a yellow from the drop down menu.
F) Click anywhere on the MOE screen to deselect the atoms. They should now appear
in the color you selected.
G) Return to the Residue Selector and click “Clear.”
H) Click the “Hydrophilic” button and then the “Select atoms” button to select all of the
hydrophilic residue atoms.
I) Highlight the atoms by putting them in “Space-filling” mode and making them the color
green.
J) Click anywhere on the screen to deselect the atoms. **A virtual slicing of the enzyme
is illustrated with the file HydroMovie ** View this movie now.
Question 5: What type of amino acid (hydrophobic or hydrophilic) is most common on the
outside/surface of acetylcholinesterase? What type of amino acid (hydrophobic or
hydrophilic) is most common in the core/interior of acetylcholinesterase? Does this
arrangement make good biological sense? Why or why not?
Question 6: According to the popular cell biology textbook Essential Cell Biology by
Alberts et al., “nonpolar (hydrophobic) side chains…tend to cluster in the interior of
the folded protein.” Provide a possible explanation for why there are some
hydrophilic amino acids on the interior of acetylcholinesterase.
K) Return the enzyme to its original form by first making sure that no atoms on the
structure are selected (selected atoms appear pink). Select the “Render” menu from
the top toolbar and select the “Stick” option.
L) Click the “Color” button from the right button bar, and select “Element” to color the
atoms according to their element. This should return the AChE to its original form.
4) Locate and view the active site of AChE.
A) First, make sure that you have returned AChE to its original state. If you are unsure,
close and re-open the AChE file to start fresh.
B) AChE has three central residues that compose its Catalytic Triad. They are SER 200,
GLU 327, and HIS 440. GLU 327 and HIS 400 activate SER 200 during the catalysis
of substrate hydrolysis (Figure 3). In order to isolate these residues, the Sequence
Editor must be used. Open the Sequence Editor by pressing the “Ctrl” and “Q”
buttons simultaneously.
C) The Sequence Editor displays all of the residues present in the chains associated with
AChE in the order that they appear on the chain. The residues are numbered for
navigational and identification purposes.
D) Locate the residues of the catalytic triad: SER 200, GLU 327, and HIS 440.
57
E) Highlight these residues. Right-click on the specific residue to open the Residue dropdown menu. Select the “Atoms” option and click “Select” to highlight the atoms of that
residue. Do this for all three of the residues of the triad.
F) Once highlighted, go to the “Render” menu on the top toolbar of the main MOE screen
and select “Space filling.” You should now be able to clearly see the triad as it is
located within the enzyme.
G) Without de-selecting the catalytic triad (selected elements appear pink), click on the
“Hide” button on the Right Button Bar, and select the “Unselected” option. This will
hide all of the residues of AChE that are not part of the triad.
H) To identify the different residues, select one atom from each of the residues to label.
Click on the “Label” button on the Right Button Bar and choose the “Residue” option.
I) The active site of AChE is the residue SER 200. Select this residue by holding the
“Ctrl” button while clicking on any atom in that residue.
J) Center SER 200 by clicking the “View” button on the Right Button Bar.
K) Close the file by going to the “File” menu on the top toolbar and selecting “Close.”
Question 7: Where does the catalytic triad fall in the three dimensional structure of
acetylcholinesterase? Is the triad on the surface or the interior of the protein? Why
does this make sense? How might substrates reach the triad?
5) Locate the internal and external choline binding sites of AChE and BuChE.
A) Open the AChE.moe file. Click the “View” button on the Right Button Bar to center the
enzyme.
B) Open the Sequence Editor by pressing the “Ctrl” and “Q” buttons simultaneously.
C) Select the internal choline binding residue (TRP 84). Render the residue into “Spacefilling” mode. This residue is located at the absolute bottom of the gorge of AChE. It
binds to the choline head of a substrate or inhibitor so that the active site can bind
and react with the molecule.
D) Select the peripheral binding site (TRP 279). Render the residue into “Space-filling”
mode. This residue is located at the top and mouth of the gorge. It can bind with
certain substrates and inhibitors to hold the molecules more tightly in the active site.
Question 8: Do you note a structural similarity in these two binding sites of AChE? Given
that many AChE inhibitors bind to both these sites simultaneously, what might you
expect these inhibitors to look like?
E) Measure the distance between the two binding sites. Click the “Measure” button on
the Right Button Bar. Next, click one of the atoms that is a part of TRP 84 and one
that is a part of TRP 279. A neon green line should appear between the atoms, with a
distance in angstroms included as a part of it.
Question 9: What is the depth of the catalytic gorge of AChE?
F) Close the AChE.moe file and open the BuChE.moe file.
G) Open the Sequence Editor.
H) Locate the internal and peripheral binding sites (The numbering of the residues of
BuChE is different from that of AChE by two. Thus, the residue TRP 84 in AChE
corresponds with TRP 82 in BuChE.).
Question 10: Is there any noticeable difference between these binding sites in AChE and
BuChE. What, if any, are they?
I) Close MOE. Choose to “Discard Molecular Data.” Re-open MOE. This step resets the
settings of MOE, making future calculations run more quickly.
58
Part 3: Substrate docking on AChE
As an example of how a substrate would
dock to the active site of AChE we will use sarin.
Sarin, a nerve agent of the organophosphate type,
was created in Germany in 1938, and its structure is
shown to the right. This nerve agent was developed
for potential use as a chemical warfare agent by the
Germans in WWII, though it was never actually
used during that war. It was, however, used by the
Aum Shinrikyo cult in a terrorist attack in the Tokyo
subway in 1995. Sarin, which is usually in gaseous
form, has no color or smell and is highly toxic. The
gas is readily spread over large areas and is easily
absorbed through the skin and inhaled.
The toxicity of sarin is due to its ability to competitively inhibit the function of the
enzyme acetylcholinesterase. The inhibition of the enzyme function leads to a build up of
acetylcholine in muscle and nerve synapses throughout the body. Exposure can create
both nicotinic (i.e. twitching, cramping, weakness) and muscarinic (i.e. eye pain,
vomiting, shortness of breath) problems. If the exposure to sarin is great, complete
paralysis or death can occur.
Serine 200 in AChE reacts with sarin by covalently bonding to its phosphate
center. The fluorine acts as the leaving group for this reaction. The sarin becomes
irreversibly bonded to the active site through a process called “aging.” This process
involves the loss of the methyl (-CH3) group of the sarin, leaving a negatively charged
oxygen atom.
1) Docking Sarin to the active site of AChE.
A) Open the file “AChEBinding.moe.” This file shows sarin in relation to the catalytic triad.
Sarin has been positioned in the manner in which it would approach and bond to
Serine 200.
B) Open the Molecule Builder. Open the “Edit” menu from the top toolbar, then select
“Build,” and then click “Molecule.”
C) On the main MOE screen, select the phosphorus atom of sarin and the single bonded
oxygen atom of SER 200. This can be done by holding the “Shift” button while
clicking the two atoms. **If asked to “Reparent the atoms into one chain,” click
“Yes.”**
D) Without de-selecting the two atoms, click on the single bond button on the Molecule
Builder. This will create the covalent bond between SER 200 and sarin.
E) Remove the fluorine atom by selecting it on the main MOE screen and then clicking
delete on the Molecule Builder. This is the form that the two molecules are in when
they initially bond with one another.
2) Aging Sarin attached to AChE.
A) Age the sarin. Select the three connected carbon atoms of sarin in the main MOE
screen and delete them in the same manner as you did the fluorine atom.
B) An extra hydrogen atom will appear attached to the oxygen atom. Delete the
hydrogen.
C) Place a negative charge on the oxygen atom by selecting the atom and clicking the “1” button on the Molecule Builder.
D) Close the Molecule Builder. The sarin is now irreversibly bound to the AChE active
site.
E) Close the file by going to the “File” menu on the top toolbar and selecting “Close.”
59
Question 11: How does the removal of the three carbon atoms from sarin effect AChE?
What processes, if any, will be effected?
Part 4: Volume of Active Site Gorge of AChE and BuChE
The catalytic gorges of acetylcholinesterase and butyrylcholinesterase have very
different volumes. This variation affects the types of substrates and inhibitors that can
enter into and bind with the active sites. Both MOE and an internet based modeling
program called CASTp can be used to view and calculate the volume of the catalytic
gorges of both enzymes.
1) View the catalytic gorge of AChE.
A) Reopen the file “AChE.moe.” This will bring up the original, unaltered
acetylcholinesterase structure. Center the enzyme by clicking the “View” button on
the right button bar.
B) Locate the main binding site. Click the “Compute” menu from the top toolbar and
select “Site Finder…” to open the Alpha Site Finder.
C) Click “Apply” to locate all of the gorges on acetylcholinesterase. A list of all of the
gorges should generate after a short time.
D) Locate the gorge that contains the catalytic triad. Do this by finding the SER200,
GLU327, and HIS440 residues in one of the numbered sites. Select the catalytic site
by clicking on it.
E) Click the “Select Atoms” button to select all of the atoms associated with the gorge.
F) Click the “Create Dummies” button. A window should pop up asking “Create dummy
atoms at alpha sphere centers?” Click “Yes.”
G) Click the “Apply” button.
H) Close the Alpha Site Finder by clicking “Close.” Do not click on the main MOE screen,
or the atoms will be deselected.
I) Click the “Compute” menu from the top toolbar and select “Molecular Surface…” to
open the Molecular Surface.
J) On Molecular Surface, click the box next to “Surface Type:” and choose
“AnalyticConnolly.”
K) Toggle on the “H and LP,” “Hidden Atoms,” and “Near Selected” options.
L) Click the box next to “Color by:” and choose “Partial Charge.”
M) Click the box next to “Neutral:” and choose “Green.”
N) Click “Apply” to create the surface of the gorge. This calculation may take a minute.
Close the Molecular Surface once the calculation is complete.
O) Insert SER200 into the gorge. Open the Sequence Editor by pressing the “Ctrl” and
“Q” buttons.
P) Locate SER200 and right click on it. Select the atoms and render them into spacefilling mode in the same manner as used for the location of the catalytic triad. Color
the residue red.
Q) Close the Sequence Editor.
R) View the gorge and active site.
Question 12: Where is SER200 located within the gorge? How could this location prevent
the binding of certain substrates?
S) Close MOE. Click “Discard Data” if prompted.
2) Determine the volume of the catalytic gorge of AChE.
A) Open Internet Explorer and go to www.Google.com. Search for “CASTp.”
B) Click the “Search CASTp Database” link. This should open the main CASTp page.
C) Click “Calculation Request” on the far left of the screen.
D) Click the “Browse” button next to the search box. Locate the AChE file [AChE
(Torpedo Californica)] and load it. The file type is a PDB file. Once the file appears in
the box, click the “Submit” button. The calculation might take a few minutes.
E) The structure of acetylcholinesterase should appear.
60
F) All of the gorges of the enzyme should appear in a list to the left side of the screen. To
view a gorge, toggle on the box corresponding to that gorge. This will also generate a
list of all of the atoms and residues present in that gorge. NOTE: viewing more than
one gorge at a time causes the program to move slowly.
G) Locate the gorge that contains SER200 and HIS440. Do this by selecting a gorge and
viewing all of the residues and atoms.
H) Once the correct gorge is located, record the volume of the gorge. This number is
located next to the gorge’s name. The volume is given in cubic Angstroms (Å3).
I) Return to the previous page by clicking the “Back” button.
Question 13: What is the approximate volume of the catalytic gorge of AChE?
3) Determine the volume of the catalytic gorge of BuChE.
A) Now load the BuChE file [BuChE (Human)] into the box. Click “Submit.”
B) Once the enzyme appears, locate the gorge that contains SER198 and HIS438.
Butyrylcholinesterase has the same catalytic triad at the base of its catalytic gorge,
but it is located two positions up in the entire primary sequence.
C) Record the volume of this gorge.
Question 14: What is the volume of BuChE’s catalytic gorge? How does this volume
compare to that of AChE? What might be the cause of any difference? What
conclusions about substrate and inhibitor specificity can be drawn from this
difference?
D) Close the Explorer window.
4) Examine the amino acid sequence to account for difference in volumes.
A) View the alignment of the amino acids of AChE and BuChE. Alignment was performed
with ClustalW. The asterisks indicate amino acid identity. The colons indicate very
high similarity, and the dots lower similarity. The absence of any mark indicates that
there is no similarity at that location in the sequence. For technical reasons,
numbering does not start at the amino terminus.
AChE
BuChE
-MNLLVTSSLGVLLHLVVLCQADDHS----ELLVNTKSGKVMGTRVPVLSSHISAFLGIP 34
MHSKVTIICIRFLFWFLLLCMLIGKSHTEDDIIIATKNGKVRGMNLTVFGGTVTAFLGIP 32
. :. .: .*: :::**
.:*
:::: **.*** * .:.*:.. ::******
AChE
BuChE
FAEPPVGNMRFRRPEPKKPWSGVWNASTYPNNCQQYVDEQFPGFSGSEMWNPNREMSEDC 94
YAQPPLGRLRFKKPQSLTKWSDIWNATKYANSCCQNIDQSFPGFHGSEMWNPNTDLSEDC 92
:*:**:*.:**::*:. . **.:***:.*.*.* * :*:.**** ******** ::****
AChE
BuChE
LYLNIWVPSPRPKSTTVMVWIYGGGFYSGSSTLDVYNGKYLAYTEEVVLVSLSYRVGAFG 154
LYLNVWIPAPKPKNATVLIWIYGGGFQTGTSSLHVYDGKFLARVERVIVVSMNYRVGALG 152
****:*:*:*:**.:**::******* :*:*:*.**:**:** .*.*::**:.*****:*
AChE
BuChE
FLALHGSQEAPGNVGLLDQRMALQWVHDNIQFFGGDPKTVTIFGESAGGASVGMHILSPG 214
FLALPGNPEAPGNMGLFDQQLALQWVQKNIAAFGGNPKSVTLFGESAGAASVSLHLLSPG 212
**** *. *****:**:**::*****:.** ***:**:**:******.***.:*:****
AChE
BuChE
SRDLFRRAILQSGSPNCPWASVSVAEGRRRAVELGRNLNCNLNSDEELIHCLREKKPQEL 274
SHSLFTRAILQSGSFNAPWAVTSLYEARNRTLNLAKLTGCSRENETEIIKCLRNKDPQEI 272
*:.** ******** *.*** .*: *.*.*:::*.: .*. :.: *:*:***:*.***:
AChE
BuChE
IDVEWNVLPFDSIFRFSFVPVIDGEFFPTSLESMLNSGNFKKTQILLGVNKDEGSFFLLY 334
LLNEAFVVPYGTPLSVNFGPTVDGDFLTDMPDILLELGQFKKTQILVGVNKDEGTAFLVY 332
: * *:*:.: : ..* *.:**:*:.
: :*: *:*******:*******: **:*
AChE
BuChE
GAPGFSKDSESKISREDFMSGVKLSVPHANDLGLDAVTLQYTDWMDDNNGIKNRDGLDDI 394
GAPGFSKDNNSIITRKEFQEGLKIFFPGVSEFGKESILFHYTDWVDDQRPENYREALGDV 392
********.:* *:*::* .*:*: .* ..::* ::: ::****:**:. : *:.*.*:
AChE
BuChE
VGDHNVICPLMHFVNKYTKFGNGTYLYFFNHRASNLVWPEWMGVIHGYEIEFVFGLPLVK 454
VGDYNFICPALEFTKKFSEWGNNAFFYYFEHRSSKLPWPEWMGVMHGYEIEFVFGLPLER 452
***:*.*** :.*.:*::::**.:::*:*:**:*:* *******:************* :
AChE
BuChE
ELNYTAEEEALSRRIMHYWATFAKTGNPNEPHSQESKWPLFTTKEQKFIDLNTEPMKVHQ 504
RDNYTKAEEILSRSIVKRWANFAKYGNPNETQNNSTSWPVFKSTEQKYLTLNTESTRIMT 502
. *** ** *** *:: **.*** *****.:.:.:.**:*.:.***:: ****. ::
61
Continues on next page.
62
AChE
BuChE
RLRVQMCVFWNQFLPKLLNATETIDEAERQWKTEFHRWSSYMMHWKNQFDHYS-RHESCA 563
KLRAQQCRFWTSFFPKVLEMTGNIDEAEWEWKAGFHRWNNYMMDWKNQFNDYTSKKESCV 562
:**.* * **..*:**:*: * .***** :**: ****..***.*****:.*: ::***.
AChE
BuChE
EL 565
GL 564
*
B) One of the features of acetylcholinesterase is that the catalytic gorge is lined with
fourteen aromatic amino acids. These are Tyr70, Trp84, Trp114, Tyr121, Tyr130,
Trp233, Trp279, Phe288, Phe290, Phe330, Phe331, Tyr334, Trp432, and Tyr442.
Locate these residues in AChE and find the homologous residues in BuChE.
Question 15: Did you find any differences between the two enzymes? If you did find a
difference can you use it to explain any volume differences between AChE and
BuChE?
Question 16: Residues Phe288 and Phe290 are particularly important as they form the
acyl pocket of AChE which is very important in substrate binding. Are their
differences between AChE and BuChE at this site? If so, how could they be used to
explain differences in substrate specificity of the two enzymes?
Part 5: Structure of cholinesterase inhibitors
Acetylcholinesterase and butyrylcholinesterase each have several different inhibitors
that affect the enzyme either competitively or allostericaly. The four inhibitors to be built
are decamethonium, BW284c51, iso-OMPA, and ethopropazine.
C
C
C N C C C C C C C C C C N C
C
C
Decamethonium
C
C C
C C C N C
C
C
C
C
C
C
C
C
C
C
N
S
C C C C C C C
C C
C
C C
C N C C C
C C
O
BW284c51
C
C
N
C
C
C
C
C
C
C
C
C
C
C
N H O H
C
C N P O P N C
C
C
Ethopropazine
C
C
H O H N
C
C
C
Iso-OMPA
**The majority of the hydrogen atoms present in these inhibitors have been left out of the above
figures. These atoms are present, and will be automatically added during the minimization
process of Spartan ’04**
63
Your instructor and T.A. will be present to answer any questions you might have about
Spartan ’04. Follow the directions below to build inhibitors of cholinesterases.
The following is a view of the main window when Spartan ’04 is opened:
The icons in the tool bar at the top are displayed below:
.
New- Opens new molecule window
Remove- Removes atoms
Open- Opens previously saved
window
Add Bond
Close- Closes current window without
closing program
Save- Saves current window
View- Finalized molecule
Add- Allows addition of new atoms to
current molecule
Remove Bond
Minimize- Calculates most stable
structure
Distance- measure length of bond
between two selected atoms
Angles- measure angle between
three selected atoms.
64
1) Build decamethonium.
A) Open Spartan ’04 by clicking on the icon on the desktop.
B) Open a new molecule window by clicking the New icon on the top toolbar.
C) Start building the inhibitor by making the ten carbon chain. Use the carbon with four
available bonds on the “Ent” tab of the Entry Model Kit.
D) Next, add the nitrogen atoms to either end of the chain. You will need to use the “Exp”
button.
tab of the Entry Model Kit. Select the nitrogen atom and then the
E) Finish the structure by adding the remaining carbon atoms to the nitrogen atoms. Use the
carbon atom that is on the “Ent” tab of the Entry Model Kit.
F) Determine the most stable conformation of the inhibitor by clicking the Minimize button
on the top toolbar. This will add the hydrogen atoms that were missing in the initial
figures.
G) Measure the length and width of the inhibitor. Do this by clicking the
button on the
top toolbar. Click on an atom at one extreme of the length of the inhibitor, and then click
on one at the other extreme. The length should appear in the bottom right hand corner of
the screen. Repeat using the width. Record this data in the table at the end of this lab.
H) Close the current molecule window, and open a new one. Do this by clicking the Close
icon on the top toolbar, and then clicking the New icon.
2) Build BW284c51.
A) Start building this inhibitor from either one of the ends. Begin with the double-bonded
carbon atoms. Then, use the carbon atom with four available bonds on the “Ent” tab of
the Entry Model Kit.
B) Next, add the nitrogen atom. Use the same nitrogen atom as before (use the “Exp” tab on
the Entry Model Kit). Return to the “Ent” tab after adding the nitrogen atom.
C) For the aromatic ring that is a part of the inhibitor, choose “Benzene” from the box next to
“Rings” on the Entry Model Kit. Click on the bond to which you want the ring bonded.
Make sure that you reselect the correct carbon atom after adding the ring.
D) When you are adding the double-bound oxygen atom, use both the carbon and oxygen
atoms with double bonds.
E) Once you have completed the entire inhibitor, determine the most stable conformation of
the inhibitor by clicking on the Minimize icon on the top toolbar.
F) Determine the length and width of the inhibitor. Record the data.
G) Close the current molecule window, and open a new one.
3) Build iso-OMPA.
A) Start building this inhibitor by using the carbon atoms with four bonds on the entry model
kit.
B) The nitrogen atoms of this inhibitor have three available bonds. Use the nitrogen atom
with three bonds on the “Ent” tab of the Entry Model Kit for this.
C) The phosphate atoms present in this inhibitor have five available bonds. Use the “Exp”
tab of the Entry Model Kit. Select the phosphorus atom (symbol “P”) and then click the
button. Return to the “Ent” tab after inserting the phosphorus.
D) The double bound oxygen atoms must be made. To do this, select the oxygen atom with
two available single bonds and attach it to the phosphorus atom. Make the double bond
by clicking the Make Bond icon from the top toolbar. Next, click the available bond on
the oxygen and an available bond on the phosphorus. Make sure that you click on the
Add icon on the top toolbar after you have made the bond.
E) Once you have completed the entire inhibitor, determine the most stable conformation of
the inhibitor by clicking on the Minimize icon on the top toolbar.
65
F) Determine the length and width of this inhibitor. Record the data.
G) Close the current molecule window, and open a new one.
4) Build ethopropazine.
A) Start building this inhibitor by adding a benzene ring to the molecule window (Use the box
next to “Rings” on the Entry Model Kit).
B) Next, add a nitrogen atom with three available bonds to one of the free bonds of the ring.
C) To one of the free bonds of the nitrogen atom, add another benzene ring.
D) Add the sulfur atom that has two single bonds to one of the ring carbon atoms that is
adjacent to the carbon attached to the nitrogen atom.
E) Connect the available bond of the sulfur to the other ring. Use the Make Bond button on
the top toolbar. Make sure that you make the bond so that the structure will look like the
figure of ethopropazine.
F) Click the Minimize icon on the top toolbar. This will make your current structure easier to
work with.
G) Continue building the inhibitor by adding the carbon atoms to the available bond of the
nitrogen atom.
H) The remaining nitrogen atom also has three available bonds. Use the appropriate
nitrogen atom on the “Ent” tab of the Entry Model Kit.
I) Once you have completed the inhibitor, determine the most stable conformation by
clicking on the Minimize icon on the top toolbar.
J) Determine the length and width of this inhibitor. Record the data.
K) Close Spartan ’04 when finished.
66
BI 125 Molecular Modeling Worksheet
Name:_______________________
Date:________________________
**This worksheet is due at beginning of next lab period.**
**It will be provided in file form, so you do not need to remove this copy from your lab manual.**
Questions 1-16: Found within the text of the Molecular and Protein Modeling exercises. Write complete
answers to each question on a separate piece of paper and staple the paper to this worksheet. Be sure that
you put your name on the attached page(s).
Question 17: Complete the following data table. Based on what you know about the structure of the binding
sites and volume of the catalytic gorges of AChE and BuChE, predict which enzyme (AChE or BuChE) will be
inhibited most effectively by each drug. Then determine the actual specificity of the inhibitors by using the
PubMed and Ester databases. From the Esther homepage
(http://bioweb.ensam.inra.fr/ESTHER/general?what=index), click on Kinetics and then Inhbitors.
Inhibitor
Length
Width
Predicted Specificity
Actual Specificity
Decamethonium
BW284c51
Iso-OMPA
Ethopropazine
67
Weeks 3-5: Enzyme Assays
CATALYSIS BY CHOLINESTERASES
For review, a simplified reaction depicting the hydrolysis of acetylcholine (Ach) is shown
below. Note that ACh is hydrolyzed into acetate and choline. In the case of butyrylcholine
(BCh), the products are butyrate and choline.
Figure 4. Hydrolysis of acetylcholine.
Notice that the reaction arrow is pointing in two directions. This is because the reaction is
theoretically reversible. In vivo, the reverse reaction happens at a low rate because acetate
diffuses away from the synapse and choline is actively taken up into the nerve terminal.
However, in vitro, under certain conditions the reverse reaction can occur and must be
accounted for in any kinetic analysis.
The ChE enzyme solution that you will use in the laboratory was made by dilution of horse
serum in sodium phosphate buffer (pH 7.0). One of the goals of the initial series of
experiments is to determine whether it is AChE or BuChE. While the ChE you will be
working with is from the horse, all vertebrate AChEs/BuChEs are similar, and all
mammalian AChEs/BuChEs are highly conserved. Thus, what is true for horse serum ChE,
whether it is AChE or BuChE can be generalized to other AchEs and BuChEs, particularly
mammalian, including human.
Quite often, natural substrates and products are difficult to assay (measure or quantify)
without laborious analytical procedures. Accordingly, biochemists often use artificial
substrates or products that are colored and therefore easy to detect and measure. For
instance, if a colored substrate were used, the reaction could be monitored by
disappearance of color as the substrate was converted to product. Conversely, the
appearance of a colored product could also be monitored. In other cases, biochemists use
coupled reactions, where the product of a reaction itself reacts with a second compound
that produces a colored product. An example of the last case is the Ellman esterase assay
(Ellman et al., 1961), which is virtually the only assay used for ChEs at the present tme. In
the Ellman assay, acylthiocholine esters, rather than acyloxycholine esters are used as
substrates. Hydrolysis of the acylthiocholine substrates (e.g. acetylthiocholine or
butyrylthiocholine) produces thiocholine which in turn reacts with a color reagent dithiobis-nitrobenzoic acid (DTNB), liberating the yellow colored 5-thio-2-nitrobenzoate ion
(Fig. 5). Each thiocholine product produced reacts with a DTNB molecule so there is an
exact one to one correspondence in the concentration of both products.
68
Figure 5. Color assay for cholinesterases. In this reaction, acetylcholine is hydrolyzed to
acetate and thiocholine. Thiocholine reacts with DTNB producing the yellow colored 5-thio2-nitrobenzoate ion.
QUANTIFICATION OF ENZYME ACTIVITY
Turn now to Appendix F: Spectrophotometry and review this section carefully, because
you must learn proper usage of the Spectronic 20 Spectrophotometer before starting this
exercise.
When a solution owes its color to a dissolved substance (a solute), the intensity of color
depends on the solute concentration. The relationship between color intensity and solute
concentration is linear and is the basis for colorimetric determinations of concentration.
Colorimetry is the quantitative measurement of color intensity with a spectrophotometer.
The spectrophotometer passes a beam of monochromatic light through a solution and, with
a photodetector, measures the proportion of light transmitted through the solution in units of
absorbance or optical density (OD). The more light that passes through, the lower the
optical density. A yellow solution absorbs light at all wavelengths except for yellow. Thus, if
we pass a beam of blue light through a yellow solution, we will record a high OD.
Conversely, a beam of yellow light would not be absorbed and we would record a low OD.
Thus, to measure the concentration of a colored solute, it is first necessary to find a
wavelength of light that is highly absorbed by that substance.
69
A spectrophotometer measures the total OD of a solution without regard for what
substances actually absorb the light. Therefore, you must adjust the instrument so that at a
given wavelength it is only sensitive to the OD of the solute in question. Your experimental
solutions will contain water, buffer, enzyme, substrate, and product. You only want to
measure the OD of the product. Refer to Appendix F: Spectrophotometry for
instructions on use of the Spectronics 20 Spectrophotometer (Spec-20).
Why is a wavelength (l) of 410 nm chosen?
A cuvette containing all of the solutes used in the experiment except the solute that you
want to measure is called the blank. The blank is used as a reference standard and all
subsequent measurements are made relative to the blank.
In order to measure an unknown concentration of a solute or dissolved product, you must
first know the extinction coefficient (ε), the proportionality constant between absorbance
and product concentration, in this case nitro-thio-benzoate.
The extinction coefficient is sometimes called the molar absorption coefficient. Every
molecule that absorbs light has a characteristic extinction coefficient at any given
wavelength. The extinction coefficient (ε) is simply the absorbance (A) divided by the
product of the concentration (c) times the length (l) of the light path (with a 1 cm cuvette
this is 1 cm) or
ε = (A/c) l
The extinction coefficient is numerically equivalent to the absorbance of a 1 M solution with
a light path of 1 cm and is expressed as M-1 cm-1.
One can determine the extinction coefficient of ONP from a standard curve by calculating ε
for each ONP concentration and averaging the result. Alternatively, the extinction
coefficient can be calculated as the slope of the standard curve. Write your result in your
notebook.
The equation ε = A/c l can be rearranged to
c = (A/ε) l.
Since the pathlength of light through a cuvette in a spectrophotometer is almost always 1
cm, the equation can be simplified to
c = (A/ε),
where ε has the units M-1.
You can use this equation to convert absorbance readings to concentration.
70
Week 3: MEASURING THE RATE OF REACTION: Ellman Esterase Assay
Work in groups of two. Store the enzyme and substrate solutions that you are given
on ice. The reason you are doing this part of the laboratory is to learn the assay.
Do not include this reaction in your laboratory paper.
1.
Turn on the spectrophotometer
2.
Use the stylus to double click on ChE on the desktop.
3.
Click on Data, then on Clear All Data.
4.
Prepare two cuvettes, each containing 0.3 ml of 2.5 mM acetylthiocholine (AsCh) and
2.4 ml of Ellman solution (0.10 M phosphate buffer, pH 7.0; 0.33 mM DTNB). Make
sure the cuvettes are very clean.
5.
To one of the cuvettes add 0.3 ml of phosphate buffer. Gently mix the contents, place
the tube in the spectrophotometer, and adjust the absorbance to 0.000, using the
directions on the preceeding page (this serves as the blank and adjusts the sensitivity
of the spectrophotometer to the range of your assay).
6.
Add 0.3 ml of the enzyme to a second cuvette, mix quickly but gently, and quickly place
it in the spectrophotometer.
7.
Click on Collect Data and record for 5 minutes, then click on Stop. You may need to
click on the clock on the menu bar to set the correct read time.
During the early part of the reaction, the product concentration increases over time in a
linear fashion. This means that the rate or velocity (Δ concentration/ Δ time; dC/dt) of the
reaction is constant. As the reaction proceeds, the rate of increase in product may diminish
and the relation between concentration and time may become non-linear. The Greek letter
delta, Δ, is used to denote “change in.”
7.
Determine the linear portion of your plot; then, position the stylus at the beginning of
the linear portion of the data, hold down the button on the stylus, and drag the stylus
over the linear portion of the graph. Lift the stylus It is possible that your reaction will be
linear throughout the experiment.
8.
Click on the R= button at the top of the screen for a linear fit of your data.
9.
Record the slope (m) in your notebook. Save the file appropriately if you wish.
This slope represents the initial rate or velocity of substrate hydrolysis as a function of
time (A/min). The initial rate is an important parameter since it is a value that does not vary
under constant conditions. Consider how altering substrate or enzyme concentration might
change the reaction rate shown in your curve. Does the rate of change in absorbance
become non-linear after a while and eventually go to zero?
71
The slope of this graph gives the intial rate as A/sec. However, we want the rate as ΔC/min
μM AsCh (or BsCh) hydrolyzed/min
This conversion is performed by multiplying by 60 sec/min and by using the extinction
coefficient for the 5-thio-2-nitrobenzoate ion:
ε= 1.36 X 104 M-1 cm-1
Remember, since the pathlength is 1 cm, we can ignore this term. Thus,
ΔC/min = (ΔA/min)/13,600 = M/min
or
ΔC/min = (ΔA/min)/13.6 = mM/min
and we will use
ΔC/min = (ΔA/min)/0.0136 = μM/min
Thus to convert your data from A/sec to μM/min, multiply the rate in
A/sec by 4.41x103.
Record the rate of hydrolysis of AsCh in μM/min in your notebook.
Experiment continues on next page.
72
Week 3 continued: EFFECT OF SUBSTRATE CONCENTRATION ON RATE OF
HYDROLYSIS I: HYDROLYSIS OF ACETYLTHIOCHOLINE
Serial Dilution of Substrate Stock Solution
You will be given a solution of 20 mM AsCh in sodium phosphate buffer. In labeled test
tubes, make a 1:2 serial dilution of the substrate as shown below to prepare stock solutions
of substrate a different concentrations.
1. Pipette 1 ml of sodium phosphate buffer into tubes S2-S8.
2. Pipette 2 ml of 20 mM AsCh into tube S1.
3. Transfer 1 ml of the 20 mM AsCh in tube 1 into tube 2. Vortex to mix. This 1:2 dilution
yields an AsCh concentration of 10 mM.
4. Transfer 1 ml of the 10 mM AsCh in tube 2 into tube 3. Vortex to mix. This 1:2 dilution
yields an AsCh concentration of 5 mM.
5. Continue for all the tubes. The last tube will contain 2 ml of solution.
6. Calculate and record the AsCh concentrations below and in your notebook.
S1
S2
S3
S4
S5
S6
S7
S8
STOCK SUBSTRATE CONCENTRATIONS
20 mM
10 mM
5 mM
_______ _________ _______ _______ ________
Store these test tubes in a rack. They do not have to be on ice.
LABORATORY CONTINUES ON NEXT PAGE
73
Assembly of Reaction
Use the serial dilution of AsCh and assemble two sets (E, Enzyme and B, Buffer) of
cuvettes (test tubes), as shown in the table below, one containing enzyme for the assays,
and one containing buffer to blank the spectrophotometer. Total volumes are 3 ml. For tube
#9, which has no substrate, use sodium phosphate buffer instead of a substrate dilution
Remember, do not add enzyme to a cuvette until you are ready to start the reaction,
and always VORTEX each tube after adding the enzyme.
FILL IN THE TABLE BEFORE PROCEEDING AND REPRODUCE IT IN YOUR
NOTEBOOK
Tube
Stock
[Substrate]
mM
ml
Substrate
Solution
ml
Ellman
Solution
ml
Enzyme
(E)
ml
Buffer
(B)
Total
Final
Volume
Final
[Substrate]
mM
1 E, B
20
0.3
2.4
0.3
0.3
3.0
2
2 E, B
10
0.3
2.4
0.3
0.3
3.0
1
3 E, B
0.3
2.4
0.3
0.3
3.0
4 E, B
0.3
2.4
0.3
0.3
3.0
5 E, B
0.3
2.4
0.3
0.3
3.0
6 E, B
0.3
2.4
0.3
0.3
3.0
7 E, B
0.3
2.4
0.3
0.3
3.0
8 E, B
0.3
2.4
0.3
0.3
3.0
0.3 (Buffer)
2.4
0.3
0.3
3.0
9 E, B
0
0
2.
Use the Ellman esterase assay to determine the rate of substrate hydrolysis at each
concentration of substrate for 2 mins. Prior to each assay, you will need to blank the
spectrophotometer to the appropriate concentration of substrate. Record the change in
absorbance over time for each reaction as done in the Ellman assay above.
3.
Determine the initial velocity (i.e. the slope) for all nine reactions (A/min) and record
these values in your notebook. There is no need to save or print the graphs. Use the
extinction coefficent for DTNB to convert the initial velocities to (μM AsCh
hydrolyzed/min) and record these values in your notebook.
4.
Next use SigmaPlot to prepare a graph showing velocity as a function of substrate
concentration, as explained on the next page.
a. Open SigmaPlot, by clicking on the icon.
74
b. In the data window, double click on the gray button 1 and enter Substrate; double click
on the gray button 2 and enter Velocity AsCh.
c. Enter the FINAL substrate concentration values into column 1 and the velocity values in
column two.
d. Click on Graph at the top of the screen, and click on Create Graph in the drop down
menu. The Graph Wizard menu will open.
e. The default graph type is Scatter Plot, which is what you want, so click on next.
f. The next default is Simple Scatter, which again is what you want, so click on next again
g. The next default is XY pair, which is again what you want, so click next.
h. The Wizard now asks for the location of the X data. Use the "Data for X " to select
Substrate Concentration.
i. Do the same for the Y data and select Velocity AsCh.
j. Click on Finish and a new window with the graph will pop up.
k. Maximize the screen and set the graph to 100% by using the "%" at the top of the
screen.
l. You will now edit your graph.
m. At the top of the graph is the default title, "2D Graph 1." Click on it and hit delete. Title of
graphs will go into the figure legends. Do the same with the key at the bottom of the
graph. Keys also go into the figure legends.
n. Double click on Y Data on the y axis and rename it Cholinesterase Activity (uM/min).
Double Click on X Data and rename it Substrate Concentration (mM).
o. Select the yellow ruler at the right side of the screen and set the Range of the x axis to
Start at 0. Do the same for the y axis if necessary.
p. To fit the data to the Michaelis-Menten equation, click on one of the data points to select
the curve to fit, then select Statistics at the top of the screen and Regression Wizard
from the drop down menu.
q. Select Hyberbola from the popup menu. The default equation is Single Rectangular,
which is the form of the Michaelis-Menten equation, y = velocity, x = substrate
concentration, a = Vmax, and b = Km.
r. Select finish.
s. Open the data window by selecting View at the top of the screen and Data 1 from the
drop down menu. Vmax is the first value in column 3 and Km is the second value. Record
these values with appropriate units in your notebook. The entries in columns 4, 5, 6, and
7 are required for the curve fit.
t. Select File and Save and save the SigmaPlot file in the location of your choice.
u. Remember, if you want to import the graph into a Word document, select the graph by
clicking on it; then copy it by using the copy icon at the top of the screen. Paste it into
Word.
5.
What are the Km and Vmax of the ChE from horse serum for the substrate AsCh?
75
Week 3 continued: EFFECT OF SUBSTRATE CONCENTRATION ON RATE OF
HYDROLYSIS II: HYDROLYSIS OF BUTYRYLTHIOCHOLINE
Serial Dilution of Substrate Stock Solution
You are repeating the last experiment; however, this time you are using the
substrate BsCh. You will be given a solution of 20 mM BsCh in sodium phosphate buffer.
In labeled test tubes, make a 1:2 serial dilution of the substrate as shown below to prepare
stock solutions of substrate a different concentrations.
1. Pipette 1 ml of sodium phosphate buffer into tubes S2-S8.
2. Pipette 2 ml of 20 mM BsCh into tube S1.
3. Transfer 1 ml of the 20 mM BsCh in tube 1 into tube 2. Vortex to mix. This 1:2 dilution
yields an BsCh concentration of 10 mM.
4. Transfer 1 ml of the 10 mM BsCh in tube 2 into tube 3. Vortex to mix. This 1:2 dilution
yields an BsCh concentration of 5 mM.
5. Continue for all the tubes. The last tube will contain 2 ml of solution.
6. Calculate and record the BsCh concentrations below and in your notebook.
S1
S2
S3
S4
S5
S6
S7
S8
STOCK SUBSTRATE CONCENTRATIONS
20 mM
10 mM
5 mM
_______ _________ _______ _______ ________
Store these test tubes in a rack. They do not have to be on ice.
LABORATORY CONTINUES ON NEXT PAGE
76
Use the serial dilution of BsCh and assemble two sets (E, Enzyme and B, Buffer) of
cuvettes (test tubes), as shown in the table below, one containing enzyme for the assays,
and one containing buffer to blank the spectrophotometer. Total volumes are 3 ml. For tube
#9, which has no substrate, use sodium phosphate buffer instead of a substrate dilution
Remember, do not add enzyme to a cuvette until you are ready to start the reaction,
and always VORTEX each tube after adding the enzyme.
FILL IN THE TABLE BEFORE PROCEEDING AND REPRODUCE IT IN YOUR
NOTEBOOK
Tube
Stock
[Substrate]
mM
ml
Substrate
Solution
ml
Ellman
Solution
ml
Enzyme
(E)
ml
Buffer
(B)
Total
Final
Volume
Final
[Substrate]
mM
1 E, B
20
0.3
2.4
0.3
0.3
3.0
2
2 E, B
10
0.3
2.4
0.3
0.3
3.0
1
3 E, B
0.3
2.4
0.3
0.3
3.0
4 E, B
0.3
2.4
0.3
0.3
3.0
5 E, B
0.3
2.4
0.3
0.3
3.0
6 E, B
0.3
2.4
0.3
0.3
3.0
7 E, B
0.3
2.4
0.3
0.3
3.0
8 E, B
0.3
2.4
0.3
0.3
3.0
0.3 (Buffer)
2.4
0.3
0.3
3.0
9 E, B
0
0
2.
Use the Ellman esterase assay to determine the rate of substrate hydrolysis at each
concentration of substrate for 2 mins. Prior to each assay, you will need to blank the
spectrophotometer to the appropriate concentration of substrate. Record the change in
absorbance over time for each reaction as done in the Ellman assay above.
3.
Determine the initial velocity (i.e. the slope) for all nine reactions (A/min) and record
these values in your notebook. There is no need to save or print the graphs. Use the
extinction coefficent for DTNB to convert the initial velocities to (μM AsCh
hydrolyzed/min) and record these values in your notebook.
6.
Next add these data to your graph show the rate of hydrolysis as a function of AsCh
concentration.
a.
b.
c.
d.
e.
Reopen the SigmaPlot file that contains the AsCh hydrolysis data.
Open the data window by selecting View and Data 1.
Select column 8 and label it Velocity BsCh.
Enter the BsCh hydrolysis values in column 8.
Then select Graph from the menu at the top of the screen and Add Plot from the drop
down menu.
77
f. The Graph Wizard menu will popup with the default Scatter Plot option, so hit Next.
g. Simple Scatter is also what you want, so hit next again, as is XY Pair, so hit next still
again.
h. Select Substrate for the X Data and Velocity for the Y Data (it will be down at the
bottom), and hit Finish.
i. Double click on a data point in the new curve and the Graph Properties menu will
popup. Change the symbol for the curve from a circle to a square by using the Symbols
Type window. Hit OK
v. To fit the data to the Michaelis-Menten equation, click on one of the data points to select
the curve to fit, then select Statistics at the top of the screen and Regression Wizard
from the drop down menu.
w. Select Hyberbola from the popup menu. The default equation is Single Rectangular,
which is the form of the Michaelis-Menten equation, y = velocity, x = substrate
concentration, a = Vmax, and b = Km.
x. Select finish.
y. Open the data window by selecting View at the top of the screen and Data 1 from the
drop down menu. Vmax is the first value in column 9 and Km is the second value. Record
these values with appropriate units in your notebook. Select File and Save and save the
SigmaPlot file in the location of your choice.
j. Remember, if you want to import the graph into a Word document, select the graph by
clicking on it; then copy it by using the copy icon at the top of the screen. Paste it into
Word.
7.
What are the Km and Vmax of the ChE from horse serum for the substrate BsCh? In the
past, a double-reciprocal or Linewearver-Burk plot was the accepted way of
determining Km and Vmax . However, with computers it is now common to perform
nonlinear regression and to fit the Michaelis-Menten equation to the data in the Velocity
vs. Substrate graph.
What kind of ChE is present in horse serum? Remember that one of the criteria used to
identify ChEs is substrate specificity as determined by Vmax ratios. Calculate the
VmaxBsCh/VmaxAsCh ratio for horse serum ChE. On the basis of this criterion is the enzyme
AChE or BuChE?
78
WEEKS 4-5: DATA ANALYSIS AND EXPERIMENTAL DESIGN FOR
CHOLINESTERASE PRESENT IN HORSE SERUM
You have learned several major procedures and concepts: how to use the spectrometer;
how to use an extinction coefficient; how to assay ChE, and how to examine the effects of an
independent variable (substrate) on a dependent variable (reaction rate).
Before beginning your experimental design, your instructor will spend some time discussing the
data from last week’s experiment and the design of your experiments. Remember that good
experimental design requires that you change only one variable at a time, while holding all others
constant in order to test the effects of the one you choose. If you look back at the earlier exercises
you will see that this condition was true.
There are several questions which arise in examining the data from the last experiment.
Answer these questions in your notebook.
1. What was the question that you were asking?
2. What is the purpose of a control measurement? Did you use appropriate controls?
3. How much confidence do you have that each rate measurement was accurate? How could
you improve your confidence?
4. Was the graph of rate vs. [S] the best representation of the data? Read the section in your
textbook on enzyme kinetics. What are the relevance of Km and Vmax? Can you determine
Km and Vmax from your plot?
_______________________________________________________________________________________
_
EXPERIMENTAL DESIGN
DETERMINING THE CHOLINESTERASE PRESENT IN HORSE SERUM
AT THIS POINT YOU WILL PREPARE A DETAILED PROTOCOL FOR DETERMINING THE NATURE OF
THE CHOLINESTERASE PRESENT IN HORSE SERUM. USE WHAT YOU HAVE LEARNED IN
PREVIOUS LABS AND IN LECTURE TO DESIGN YOUR EXPERIMENT. YOUR TA AND INSTRUCTOR
WILL BE PRESENT TO HELP YOU WITH THIS PROCESS.
79
LABORATORY REPORT
Members of a group should pool and discuss their data; however, each individual must write his/her
own report. This report is to cover all the experiments performed in the enzyme exercises over the last
five weeks, including molecular modeling, effect of substrate concentration on velocity, substrate
specificity, nature of ChE from horse serum, and experiments that were designed.
For further basic background information see any good biochemistry, molecular biology, and/or genetics text
book, and consult your own text. More importantly, consult the articles on research and search for
additional review articles and primary research articles in the library.
REFERENCES CITED (Additional Papers are on reserve)
Chatonnet, A. and O. Lockridge (1989) Comparison of butyrylcholinesterase and acetylcholinesterase.
Biochem. J. 260, 625-634.
Ellman, G.L., D. Courtney, V. Andres and R.M. Featherstone (1961) A new and rapid colormetric
determination of acetylcholinesterase activity. Biochem.Pharm.7, 88-95
Massoulié, J., L. Pezzementi, S. Bon, E. Krejci and F-M. Vallette (1993) Molecular and cellular biology of
cholinesterase. Pregress in Neurobiology 41, 31-91.
Some Suggestions for the ChE Paper, Biology 125, Cell and Molecular Biology
Birmingham-Southern College, Leo Pezzementi
Introduction
AChE and BuChE characteristics, ways of distinguishing: Vmax ratio, substrate inhibition,
diagnostic inhibitors. Don’t spend a lot of time on general enzyme information.
What is reported in the literature concerning the nature of the ChE in horse serum and
other vertebrate sera? Find multiple relevant sources, not just those on reserve.
Do not discuss the Ellman assay.
Materials and Methods
In Materials, just list the materials.
It is not necessary to give the concentrations of substrate for the substrate specificity
experiments, since they can be obtained from the graph; likewise for the inhibitor
concentrations in the inhibition experiments. However, it is necessary to give final substrate
concentrations in the inhibitor experiments.
Combine protocols for acetylthiocholine and butyrylthiocholine hydrolysis experiments.
Do not give number of test tubes used in an experiment.
Do not describe how to make a serial dilution.
Include wavelength used, final DTNB concentration in assay and its extinction coefficient.
80
Results
Use your data for the Michaelis-Menten graph; class data for the Km and Vmax.
Use class data for dose-response curves and for IC50 values.
Give brief description of experiment and indicate how data were fit in figure legends of
appropriate graphs.
Don’t forget to include results from molecular modeling.
Discussion
Overview of results.
Kinetics, Vmax ratio, substrate inhibition. Explain the molecular basis citing multiple sources
in the literature.
Compare and contrast your data with multiple sources in the literature concerning the ChE
in horse serum. Do not cite laboratory manual
Diagnostic inhibitors. Explain the molecular basis and cite multiple sources in the literature.
Do not cite laboratory manual.
References
Use all papers on reserve in library and others.
Do not use online sources unless they are refereed publications that are available online.
You must attach a copy of the front page of every reference that you use. If you
download an article from the internet, print out the PDF version of the article, not the
html version.
Shown below is the homepage address for the Cholinesterase database. Do not cite this
page, but use it as a source of references. The “Kinetics” section of the webpage is
especially helpful with information regarding inhibitors of cholinesterases.
http://bioweb.ensam.inra.fr/ESTHER/general?what=index
81