ENZYME KINETICS —Acetylcholinesterase
Biol 303 — Comparative Animal Physiology
Summary:
(1) Follow the progress of an enzyme catalyzed reaction by measuring the amount of colored end product spectrophotometrically as a function of time.
(2) Apply Beer’s Law and a known extinction coefficient to the data from the reaction progress plot (step 1) to determine the initial reaction rate in appropriate chemical units, mM/min.
(3) Run a series of reactions, each time measuring initial reaction (as done in step 2), to study how the rate varies with substrate and enzyme concentration and with temperature.
(4) Use the data collected in step 3 to determine the properties of the enzyme: Km. vmax, Specific Activity, and Q10.
Report:
The following items are required: Note that a "working" graph is intended for your own use, not fr publication. It must have data correctly plotted, with correct numerical values and units on each axis, but may include working lines and calculations and may be missing a caption. A "proper" graph is a formal presentation to show to others, neatly drawn and aesthetically proportioned, with informative axis labels and caption and no extraneous marks or scribbles.
(1) A series of "working" graphs illustrating absorbance vs. time for each of your reactions showing how initial reaction rates are calculated.
(2) Two "proper" graphs, one showing the relation between reaction rate and substrate concentration, the other showing the relation between reaction rate and enzyme concentration.
(3) A "working" Lineweaver-Burke plot of your data, illustrating the calculation of the enzyme.
(4) A "proper" statement, in one or two sentences, presenting the numerical values for the properties of the enzyme: that is, Km, vmax, specific activity, and Q10.
You will be graded, in part, on the accuracy of your work. You will not be held responsible for any problems that result from problems with the supplies or equipment outside your control provided you bring these problems to the attention of your instructor during the lab period.
Biol 303 Enzyme Kinetics – AChase Part I p. 2
Background
Acetylcholinesterase (AChE) is an important enzyme in the nervous system. Acting at the synapse, it hydrolyzes the neurotransmitter acetylcholine (ACh) to acetate plus choline. This inactivation of the transmitter helps terminate the synaptic response. AChE is one of the most efficient enzymes known with a turnover time of 150 msec. That is, one molecule of AChE can hydrolyze about 6000 molecules of Ach in one second.
In order to follow the progress of any enzyme catalyzed reaction, it is necessary to make continuous or repeated assays of the concentration of one of the reaction substrates or products. If one of these substances is colored this assay is quite simple. The light absorbance of the reaction mixture is measured photometrically and changes in the absorbance are interpreted as changes in the concentration of the colored substance according to Beer's law, summarized below. You should review the principles of spectrophotometry and the operation of the Spectronic 21 spectrophotometer before coming to lab. The Science Learning Center has modules on these topics that you can review.
See: http://www.umd.umich.edu/casl/natsci/slc/slconline/SPC21/
In this experiment, neither the substrate nor either product is easily measured, so an alternate reaction is used, where the artificial substrate acetylthiocholine iodide (ATCh) is hydrolyzed into acetate and thiocholine. The sulfhydryl group in thiocholine reacts with dithiobisnitrobenzoate (DTNB) to produce two compounds, 2-nitrobenzoate-5-mercaptoathiocholine and 5-thio-2-nitrobenzoate. The latter compound has a yellow color and can be measured in a spectrophotometer. The second reaction is rapid and complete. Therefore, the concentration of the final colored end product is a direct measure of the concentration of thiocholine produced.
A concentrated dye solution is darker – absorbs more light – than a dilute solution. This is expressed mathematically by Beer’s Law: A = e C d, where A is the light absorbance, measured by the spectrophotometer, and C is the concentration of dye. The proportionality constant, e, is called the extinction coefficient and d is the path length, the distance the light has to travel through the solution. We will measure light absorption at 412 nm wavelength, using cuvettes with a 1 cm path length. The extinction coefficient for 5-thio-2-nitrobenzoate is 13,600 M-1 cm-1.
You will be running a series of enzyme reactions by mixing enzyme (AChE), substrate (ATCh) and color developer (DTNB) in different concentrations. As the reaction proceeds, ATCh is hydrolyzed and the product reactions with DTNB to produce another compound with a yellow color. By measuring the absorbance as a function of time, always at 412nm, and using the known extinction coefficient for the colored end product, you can determine the concentration of product as a function of time.
First you will first run a series of reactions using a constant amount of enzyme, but varying how much substrate you use. Then you will run a series of reactions varying the amount of enzyme, but keeping substrate concentration constant. Finally you will run the reaction at two different temperatures keeping both enzyme and substrate constant. You must make sure that all the reactions have the same amount of DTNB and the same total volume. In order to keep the total constant while varying either enzyme or substrate, you use a neutral “filler” substance to make up the total volume. As you increase the amount of one substance, you compensate by decreasing the amount of filler. For this experiment, the filler is a phosphate pH buffer at pH 8.0. Note: the substrate is dissolved in the same pH buffer, not in distilled water. The color developer, DTNB is dissolved in a similar buffer, but at pH 7 – it is slightly unstable at pH 8.
The enzyme reaction
Materials
You will have available stock solutions of
· 0.1 M phosphate buffer, pH 8.0
· 0.01 M DTNB (dithiobisisonitrate, the color developer)
· 1.5 mM ATCh (Acetylthiocholine, the substrate)
· AchE (the enzyme). You will be told the enzyme concentration in lab
Blanking
Turn on the Spectronic 21 spectrophotometer and allow it to warm up at least 15 minutes. (If you have not reviewed these instructions before coming to lab, you must now pay the penalty of waiting for the machine to stabilize) Adjust the wavelength scale to 412 nm
Prepare an enzyme blank by adding 3.0 ml of pH buffer into a cuvette. Add 0.1 ml DTNB and 0.2 ml enzyme. This is called an enzyme blank because it contains all the material used in the reaction including the enzyme, but not the substrate. Use this solution to adjust the instrument to zero on the absorbance scale (100% transmission = 0 absorbance, the zero on the right). There is no reason to record percent transmission (%T) during this experiment. Keep this blank for the rest of your work. Use it periodically to verify that the spectrophotometer has not drifted away from its original setting. If so, just reblank it.
The blank must contain DTNB because it may have a slight yellow color before any reaction occurs. We use the enzyme blank rather than the more common substrate blank (containing substrate but no enzyme) because DTNB reacts with any sulfhydryl group to form a colored end product. If the enzyme contains –SH groups, it will produce some yellow color.
When you change the enzyme concentration later during the lab, you must also change the blank in a corresponding way.
The first reaction
For your first reaction, pipette 1.0 ml of substrate, 2.0 ml of buffer, and 0.1 ml of DTNB into a cuvette. Do not add any enzyme yet. The reaction begins as soon as you add the enzyme. Since you are interested in the initial rate, you must be prepared to collect data from the start. One member of the lab team should be in charge of doing the pipetting and manipulating the equipment; another should be responsible for coordination and data collection.
Add 0.2 ml of enzyme stock to the reaction mixture, recording the exact time. Quickly mix the solution and insert the cuvette into the spectrophotometer. Be fast about it! You must be able to record data starting 30 seconds from the time the enzyme first hits the reaction mixture! Record absorbance every thirty seconds for three minutes. Plot a rough sketch of absorbance vs. time in your lab notebook. Does the absorbance increase linearly with time? Can you calculate a good reaction rate by measuring the slope of the absorbance vs. time graph? If not, something is wrong. In that case, you must try the reaction again.
The initial reaction rate
The reaction rate has units of concentration divided by time. Reaction rates are traditionally measured in mM/min. The slope of the reaction curve you just plotted has units of Absorbance divided by time. How do you calculate the "real" reaction rate from your data? Use Beer’s law to make this calculation.
What controls reaction rate?
If you use more substrate, the reaction should go faster, if you use less, the reaction should go slower. If you use more enzyme, the reaction should go faster, if you use less, the reaction should go slower. If the reaction is warm, it should run faster, if it is cold, it should run slower. Run a series of reactions, first systematically varying the substrate, then systematically varying the enzyme, finally varying temperature. Each time, measure the initial reaction rate.
As you plan your work, you should keep the total reaction volume constant at 3.3 ml. You should also keep the amount of DTNB constant at 0.1 ml.
You must have data for at least five different substrate concentrations over a wide range. The most concentrated should be greater than 1 mM and the least concentration should be no larger than 0.1 mM. You replicate your work: at least two reactions at each substrate concentration. You must have data for at least three different enzyme concentrations. The highest concentration must be at least four times the lowest concentration. Again, you must replicate your work: at least two reactions at each enzyme concentration. You must have data for at least two temperatures. The highest temperature must be at least 10 °C higher than the lowest with at least two replicates. You can “overlap” the data, so that one of the reactions can be used in all three studies: variation in substrate, in enzyme, and in temperature. In all, you need to run at least 16 reactions. Your initial reaction will count as one of these. Since each reaction must run three minutes, this will take at least 48 minutes of actual reaction. With careful planning and efficient teamwork, you should be able to complete this work easily in less than two hours.
Data reporting
For every reaction you run, you will have to calculate the initial rate from the slope of your reaction progress plots (absorbance vs. time). These are “working” plots, and will probably include construction lines and miscellaneous scribbles and notes. You must turn in these plots as part (1) of your report. If you calculated initial rate some other way, for example by having a computer calculate the slope from your data tables, then turn in some form of work to indicate how you calculated initial reaction rate. You do not have to make these graphs “pretty” in any way. They must merely be accurately plotted.
You will have data from at least ten different reactions all run at the same enzyme concentration and temperature but differing in substrate concentration. Plot a “proper” graph of reaction rate vs. substrate concentration for this data. You must have proper chemical units on each axis: mM/min for reaction rate and mM or mM for substrate concentration. This plot must have a proper title and caption and be of “publishable” quality and style. This is the first graph required for part (2) of your report.
You will have data from at least six different reactions all run at the same substrate concentration and temperature but differing in enzyme concentration. Plot a “proper” graph of reaction rate vs. enzyme concentration for this data. The reaction rate must have units of mM/min and the enzyme concentration mg protein/ml. This plot must have a proper title and caption and be of “publishable” quality and style. This is the second graph required for part (2) of your report.
You will have data from at least four reactions all run at the same substrate and enzyme concentrations but differing in temperature. You do not have to plot this data. Just average the rates at each temperature and use the data to calculate a Q10 for the enzyme.
Calculating Enzyme parameters
Calculating Km and vmax
Plot a Lineweaver-Burke (double reciprocal) plot of the data from the ten reactions all done at the same enzyme concentration and temperature but varying in substrate concentration. This is very simple if you used Microsoft Excel to plot the standard linear plot of v vs [S] for part (2) of the report. You already will have two columns of data, one with [S] (substrate concentration), the other with v (reaction rate). Use the software to create two new columns, one with 1/[S], the other with 1/v. Plot this new data – your Lineweaver-Burke plot and have the computer draw a straight line through the data (linear data fit) and print out the equation for the line. If you write the equation in the form Y = m X + b (where y = 1/v and X = 1/[S]), then vmax = 1/b and Km = m/b. The units for vmax are exactly the same as the original units for v, probably mM/min and the units for Km are exactly the same as the original units for [S], probably mM.