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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