Catalase Kinetics

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CATALASE KINETICS
The Determination of Various Biochemical and Biophysical Constants for This Enzyme

OVERVlEW of the LABORATORY EXERCISE

..... In this laboratory you will observe the conversion of hydrogen peroxide (H2O2) to water and oxygen gas by the enzyme catalase. .. You will then measure the amount of oxygen generated and calculate the rates of the enzyme-catalized reactions under various conditions, along with values indicating just how voracious catalase is and what the activation energy is for the catalyzed reaction. .. Time required:.. 80 minutes for the slowest group, including assembling the necessary ring-stands, waterbaths, etc.


OBJECTlVES

Before doing this laboratory you should understand:

After doing this laboratory you should be able to:

lNTRODUCTlON

.....In general, enzymes are proteins produced by living cells; they act as catalysts in biochemical reactions. ..A catalyst affects the nte of a chemical reaction. ..One consequence of enzyme activity is that cells can cany out complex chemical activities at relatively low temperatures.

.....In an enzyme-catalyzed reaction, the substance to be acted upon (the substrate = S) binds reversibly to the active site of the enzyme (E). ..One result of this temporary union is a reduction in the energy required to activate the reaction of the substrate molecule so that the products (P) of the reaction are formed.

.....In summary:

E+S -> ES -> E+P

.....Note that the enzyme is not changed in the reaction and can be recycled to break down additional substrate molecules. ..Each enzyme is specific for a particular feaction because its amino acid sequence is unique and causes it to have a unique three-dimensional structure. ..The active site is the portion of the enzyme that interacts with the substrate. so that any substance that blocks or changes the shape of the active site affects the activity of the enzyme. ..A description of several ways enzyme action may be affected follows.

  1. Salt concentration. If the salt concentration is close to zero, the charged amino acid side chains of the enzyme molecules will attract each other. ..The enzyme will denature and form an inactive precipitate. if, on the other hand, the salt concentration is very high, normal interaction of charged groups will be blocked. new interactions will occur, and again the cnzyme will precipitate. ..An intermediate salt concenuation such as that of human blood (0.9%) or cytoplasm is the optimum for many enzymes.
  2. pH. pH is a logarithmic scale that measures the acidity or H+ concentration in a solution. ..When the pH is in the range of under 7, a solution is said to be acidic; if the pH is 7, the solution is neutral: and if the pH is in the range of over 7, the solution is basic. ..Amino acid side chains contain groups such as -COOH and -NH2, that readily gain or lose H+ ions. ..As the pH is lowered an enzyme will tend to gain H+ ions, and eventually enough side chains will be affected so that the enzyme's shape is disrupted. ..Likewise, as the pH is raised, the enzyme will lose H+ ions and eventually lose its active shape. ..Many enzymes perform optimaly in the neutral pH range and are denatured at either extremely high or low pH's. ..Some enzymes, such as pepsin, which acts in tht human stomach where the pH is very low, have a low pH optimun.
  3. Temperature. Generally, chemical reactions speed up as the temperature is raised. ..As the temperature increases, more of the reacting molecules have enough kinetic energy to undergo the rcaction. ..Since enzymes are catalysts for chemical reactions, enzyme reactions also tend to go faster with incrcasing temperature. ..However, if the temperature of an enzyme-catalyzad rcaction is raised still further, a temperature optimum is reached; above this value the kinetic energies of the enzyme and water molecules are so great that the conformation of the enzyme molecules is disrupted. ..The positive effect of speeding up the reaction is now more than offset by the negative effect of changing the conformation of more and more enzyme molecules. ..Many proteins are denatured by temperatures above 40-50°C (but some are still active at 70-80°C, and a few even withstand boiling.
  4. Activators and Inhibitors. Many molecules other than the substrate may interact with an enzyme. ..If such a molecule increases the rate of the reaction it is an activator, and if it decreases the reaction rate it is an inhibitor. ..These molecules can regulate how fast the enzyme acts. ..Any substance that tends to unfold the enzyme, such as an organic solvent or detergent, will act as an inhibitor. ..Some inhibitors act by reducing the -S-S- bridges that stabilize the enzyme's structure. ..Many inhibitors act by reacting with side chains in or near the active site to change its shape or block it. ..Many well-known poisons such as potassium cyanide and curare are enzyme inhibitors that interfere with the active site of critical enzymes.



C A T A L A S E

.....Catalase is nearly ubiquitous among organisms that can grow in the presence of oxygen (air). ..The major function of catalase within cells is to prevent the accumulation of toxic levels of hydrogen peroxide formed as a by-product of metabolic processes - primarily that of the electron transport pathway. ..The only exceptions are the "lactic acid bacteria," which cannot synthesize the fundamental building block porphyrin, and hence do not even possess cytochromes that would otherwise make the toxic H2O2.

.....Each molecule of catalase has four polypeptide chains, each composed of more than 500 amino acids, and nested within this tetrad are four porphyrin heme groups - very much like the familiar hemoglobins, cytochromes, chlorphylls and nitrogen-fixing enzymes in legumes. ..(Catalase may also take part in some of the many oxidatic reactions that occur in all cells.)

pathway by which the ETOP reduces oxygen to peroxide, which catalase then attacks

.....In the absence of catalase, this reaction occurs spontaneously, but VERY slowly. ..Catalase speeds up the reaction rate many thousands of fold. In today's experiment, a rate for this reaction will be determined.

.....Much can be learned about enzymes by studying the kinetics (particularly the changes in rate) of enzyme-catalyzed reactions. ..For example, it is possible to measure the amount of product formed, or the amount of substrate used, from the moment the reactants are brought together until the raction has stopped.

.....If the amount of oxygen formed is measured at regular intervals and this quantity is plotted on a graph, a curve like the one that follows is obtained.

Graph of oxygen formed versus seconds, showing a possible lag and then how initial velocity is determined.

.....Study the solid line on the graph of this reaction. ..At time O there is no product. Product only starts accumulating when the system has begun working. ..In this graph there is shown a lag, which may be caused by several factors. ..One is that for a few seconds any produced oxygen is dissolving into and saturating the water. .. Only after the water has become saturated does gas start coming out of solution, to fizz and be collected. ..Another factor is how long it takes for H2O2 to diffuse into the cell and then for oxygen to diffuse out. ..Thus on this graph, it took 10 seconds for gas to start being produced. ..After 15 seconds, 25 millimoles (mmoles)* have been formed; after 20 seconds, 50 mmoles; after 30 seconds, 100 mmoles. ..The rate of this reaction could be given as 5 mmoles of product formed per second for this initial period. ..Note, however, that by the 40th and 50th seconds, only a few, if any, additional mmoles of oxygen have been formed. ..After the lag and then during the first thirty seconds, the rate is constant... From thereon, the rate is changing; it is slowing down. .. For each successive period time after the 30 seconds, the amount of product formed in that interval is less than in the preceding 5 seconds. ..From the second half-minute onward, the reaction rate is very slow and stopping asymptotically.

.....Therefore the rate of the reaction is the slope of the linear portion of the curve. ..To determine a rate, pick any two points on the straight-line portion of the curve. .. Divide the difference in the amount of product formed between these two points by the difference in time between them. ..The result will be the rate of the reaction which, if properly calculated, can be expressed as mmoles product/sec. ..The rate then is:

A figure shows how to determine the rate of a reaction: delta-Y over delta-X.

.....In comparing the kinetics of one reaction with another, a common reference point is needed. .. For example, suppose you wanted to compare the effectiveness of catalase obtained from potato with that of catalase obtained from liver. ..Using known amounts of enzymes, it is best to compare their reactions when each of their rates are constant. ..In the first moments of an enzymatic reaction such as this, the number of substrate molecules is usually so large compared with the number of enzyme molecules that changing the substrate concentration does not (for a short period at least) affect the number of successful collisions between substrate and enzyme. ..During this early period, the enzyme is acting on substrate molecules at a nearly constant rate. ..The slope of the graph's line during this early period is called the initial rate of the reaction. ..The initial rate of any enzyme-catalyzed reaction is determined by the characteristics of the enzyme molecule. ..It is always the same for any enzyme and its substrate at a given temperature and pH. ..This also assumes that the substrate is present in excess.


* 100 ml of any gas at standard temperature and pressure is equal to 4.46 millimoles.



SETTING UP THE EXPERIMENT: RANGE-FINDING

.....Before beginning with experimentation, we must first invent a device for measuring the amount of oxygen gas that is produced. ..The most direct "trick" for doing this is to trap the evolving under a container that is filled with water. ..After inventing such a device, we then must determine the amounts of the enzyme and substrate that will yield workable values in that device. ..Thus, we will invent an equipment setup such that 100 to 500 ml of gas can be collected and its rate of production can be quantitatively measured. ..Then we must find amounts of yeast or gingerroot juice, which is our catalase source, and H2O2 that will produce 100 to 500 ml of O2 at a rate that is neither too fast nor too slow for our convenience. ..(Previously, it was found that the following produced about 560 ml of gas: ..20 ml of "20 volume" H2O2 + 10 ml of 5% yeast + 120 ml water.) .. Purchaser: ..Yeast should be the moist cakes of yeast. ..One cake is more than sufficient for one group's many experiments. .. "20-volume H2O2" is made by dilution "30- or 40-volume H2O2", which can be purchased from your nearest beauty supply store for about $5 per liter. ..20-vol is found to be safe for fingers.

  1. Make a 10% slurry of wet-cake bakers yeast in water. ..Weigh the cake, and then crumble it in the fingers and add it to 9-times that much water. .. Be sure to mix well to obtain a homogeneous suspension. (Alternatively, we would start with the filtrate of ginger root blended in water.)
  2. Set up the device shown below using a ring stand and clamps, of course to support a 250 or 500 ml cylinder that is graduated all the way to the bottom. .. Make sure that the tube that runs up inside the cylinder is tipped with a little piece of tubing to act as a bumper. ..Also cut a couple of SMALL notches in the bumper to allow the gas easy exit from the tube in event it is pressed tightly up against the bottom of the cylinder. .. It is strongly recommended that thin copper tubing be used where sharp bends are needed so as to eliminate most breakage during the shaking that is needed to be done. ..Regarding the reaction vessel: the tube that passes through the stopper should protrude far enough below the bottom of the stopper so that a suction tube can be attached to facilitate drawing water up the cylinder to the top during priming. ..(Once the water reaches the top, it usually stays there without having to pinch the flexible tubing after removal of the suction. .. If mechanical suction is not available, mouth suction applied through an intervening small "filter" flask with side connection is more than adequate.
  3. To the reaction vessel add 120 ml of water, 10 ml of the yeast slurry and 20 ml of the "20-volume" H2O2. We'll call this 120-10-20 mix the "std am'ts".
  4. Immediately attach the reaction vessel to the gas measuring device and begin vigorously shaking the reaction vessel.
  5. If, after 5 minutes of shaking the reaction vessel, the amount of evolved gas lies outside of 100 to 500 ml, adjust the volume of H2O2. Most "control" reactions reach completion well within 5 minutes. ..For your purposes, "completion" means that the rate of gas evolution is no longer linear. ..Remember: all you want are data points at the beginning that lie on a straight line. ..Once you have 6 to 10 points on a line, QUIT! .. Don't waste time. .. Move on to the next run. .. Hence: ..don't merely write down lists of numbers: GRAPH THEM AS YOU GO!

A diagram of a set-up required for collecting the oxygen gas that has been evolved from the catalase reaction.



KINETICS EXPERIMENTS

.....This is a real test of team-work! .. It is best to have four people in the group: "A" shakes the reaction vessel and give a shout whenever another 25 or 50 ml of gas has been evolved; "B", who watches the clock, shouts the time (usually in seconds); "C" records the time, and "D" graphs the value as shown in the figure below. .. (Taking values of equal intervals of time might seem more reasonable, but reading the cylinder is harder than reading the clock when the system is moving rapidly.) .. "D" finally announces "Stop!" when enough points are lying on a straight line. .. While "A" and "B" set up for another run at a different concentration or temperature, "C" and "D" determine the Vo (initial velocity) and neatly record that value along with its parameters. ..To determine Vo, plot "volume of evolved O2" versus "time." ..Use a ruler to draw the "initial slope" on your plot. ..The slope of that line in ml/minute is Vo. ..You must make a new plot like the one below for each "run."

Graph of oxygen formed versus seconds, showing a possible lag and then how initial velocity is determined.

  1. EVERY group does this "CONTROL:" Put the "std am'ts" into the reaction vessel, attach to the measuring device, and, while shaking the vessel vigorously, record the volumes of evolved gas at timed intervals. ..(Start the timing when the first bubble of gas reaches the graduated cylinder.)
  2. As variations on the Control, three types of experiments will be done. .. Each group should do only one of these three experiments. ..To best prepare yourself, first look at what you want your final graph to look like (see your appropriate part of #3, below). ..Each consists of several data points. ..Each such data point on your final graph is the Vo determined from a making preliminary graphs such as you did for the "control." ..(In other words: ..each group will make four to six graphs of each of their "runs," and data calculated from those will be used to make a final graph.) ..All groups will then post their results and final graphs. .. ALL participants should understand the results of ALL other groups!
    The GROUPS are:
    • Group "Delta Enzyme:" ..Vary the proportion of yeast to water (sum of the two equals 60 ml). (H2O2 is the standard amount)
    • Group "Delta Substrate:" .. Vary the proportion of H2O2 to water (sum of the two equals 65 ml). .. (Yeast is the standard amount)
    • Group "Delta Temperature:" ..Vary the temperature of the reaction by keeping the reaction vessel in a dishpan of water that has been adjusted to a given temperature. .. Use 120 ml of that temperature-adjusted water to fill the reaction vessel so as to facilitate faster temperature equilibration. ..This group should consist of students who are good in math! ..They should be able eventually to explain why it is NOT necessary for them to convert their data to mmoles/second and can use whatever units they wish (buckets per century - anything!) for their determination of activation energy. ..Supplemental reading for this group: ..Who was Arrhenius?

  3. . Each group makes one of the following three FINAL GRAPHS:

    • Group "Delta Enzyme": Vo versus relative yeast concentration (holding std H2O2 constant) You should get a straight line: Vo = m [yeast]. (m=slope)

      Presentation of results: .. Neatly draw a final graph with properly labelled axes showing the units. .. Also show the value of the slope. .. It should appear similar to this:

Vo vs am't of yeast used.

This is the most obvious result! If you look at the supermarket analogy at the end of "b" (next), your data will say that to have more check-out persons will allow the store to check out more people per hour.

    • Group "Delta Substrate": ..This group actually is to make two graphs:
    Michaelis-Menton Plot.Lineweaver-Burke Plot.
    • Vo versus [H2O2] (holding [yeast] constant) Km = [H2O2] at 0.5(Vmax); where Km is in the same units as the H2O2

    • 1/Vo versus 1/[ H2O2] (should give a straight line) A better way to find Vmax is to use this Michaelis-Menton style of graph.

      .....Km is an index of the affinity of the enzyme for the substrate.

    .....Questions for the "Delta Substrate" Group: (1) How would Km and V be affected if half of the enzyme molecules were denatured? (2) How would Km and V be affected if you added an equal concentration of something that looked very much like the substrate, and that could reversibly bind to the enzyme's active site, but that the enzyme could not split (a competitive inhibitor)?

    .....ANALOGY: A way to consider the affects of various sorts of inhibitors is to think of a supermarket. ..(1) A competitive inhibitor would be someone who enters the checkout line without money. ..The clerk tallies the items, only to discover the impecunious state of the false customer, who then must be evicted, with no useful work done. ..If half of the customers were without money, the clerks would work half their time needlessly. ..If the proportion of paying customers is increased, the efficiency of checkout is increased. .. If the proportion of payers to cashless customers approaches infinity, then greater and greater efficiency is reached. .. If the absolute number of payers reaches infinity, then Vmax is reached because the clerks are working without any lapses between the customers. ..(2) Now consider customers whose shopping carts get jammed in the checkout lane - effectively shutting down the lane. .. Does increasing the proportion of normal customers help the efficiency? .. (3) What if the teamsters go on strike and the supermarket is only sporatically stocked. ..What affect on efficiency occurs as customers increase? .. So long as the customers are few, the laggardly stocking of the shelves suffice. .. But as the customers increase, shortages rapidly become evident. ..(4) What about customers who come in with counterfeit money that is not recognized immediately? .. It would be like E. coli's trying to grow on a mix of lactose and ONPG. Whenever it grabs ONPG it puts it through the "checkout lane" alright, but then the product cannot be used. ..It might also be like customers who cannot fit out the exit door!

    .....The use of these enzymological Michaelis-Menton kinetic experiments with various types of inhibitors is useful in discovering how particular enzymes work or how poisons work.

  1. Group "Delta Temperature":.. You are to make a graph to find the ACTIVATION ENERGY:

    1. Run the "std am'ts" at various temperatures. (Try temperatures between 7°C and about 50°C)
    2. Plot ln (Vo) versus 1/T°K ; where 0°K = -273°C
    3. Eact is proportional to the negative of the slope

    Arrhenius Plot:  Ln(initial V) versus 1/Kelvin giving a line. Eact is -2x slope.

    .....An ANALOGY for activation energy might go this way: Imagine a barracks of troops. .. It is a very cold winter morning. .. The troops don't want to get out of their warm beds. .. As spring progresses however, the sergeant needs to make less and less threatening remarks to get the troops "activated." .. However, if global warming eventually made it too hot, the troops would die, and could never be "activated."

    .....Or maybe you'll like this EXAMPLE (not an analogy!) of activation energy better. .. You are at a large amusement park and have just been seated in a roller coaster. The brake is released that the train rolls foreward just a little bit. .. Ahead of you is a huge incline up which a chain pulls you to the top. .. What has happened here? .. What do you have as evidence? .. You and the train have been "activated". .. If the train were to have stalled at the very top, you would have time to look around and see that you were higher than any other peak on that ride. .. So were this a frictionless world, once released you'd "fly" all the way to the end of the line. .. But this is not a frictionless world, and so there is often another "activation" along the way. .. Happily for physicists, activation energy can often be easily calculated quite directly - what mass had to be lifted to what height to give the required potential energy.


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