Lab 4 Enzyme kinetics (1)

Lab 4: Enzyme Kinetics: Characterization of The Enzyme Alkaline Phosphatase Background on enzyme kinetics The activity of enzymes is crucial for the proper functioning of cells. Enzymes catalyze most reactions in metabolic pathways in the context of energy flow in living organisms. Not only do enzymes make most reactions possible in an intracellular environment, but enzymes also allow for the control and stabilization of these reactions. It is not a stretch to say that life itself would be impossible without enzymes. Enzymes are proteins that work at very specific environmental parameters such as temperature, pH, availability of small molecules as cofactors, or substrate concentration. The behavior of enzymes in response to different concentrations of the reaction chemicals (both substrates and products) comprise the essential characteristics of each type of enzyme. This behavior, called enzyme kinetics, is responsible for much of the reaction control in biological systems. To learn about enzymes and enzyme behavior, we will examine the kinetics of the enzyme alkaline phosphatase in this lab. Alkaline Phosphatase Enzyme Alkaline phosphatase is a ubiquitous enzyme that can be isolated from bone, kidney, intestine, plasma, liver, spleen, plants, and microorganisms. It catalyzes the removal of a phosphate attached to an alcohol, generating a free phosphate (P i ) and an alcohol. Alkaline phosphatase can act on a variety of specific substrates; the generalized reaction is shown below: O P O O - O - R + H 2 O R O H + - O P - O O - O substrate alcohol phosphate Interestingly, the precise biological function of the enzyme is unknown. Clinical interest in alkaline phosphatase was inspired by the observation that certain pathological conditions, such as obstructive jaundice, rickets, and other bone disorders, were characterized by significant increases in the plasma concentration of the enzyme. It is particularly abundant in tissues that are involved in the transport of nutrients. The fact that it is localized at the surface of absorptive tissues suggests a role in transporting nutrients across the epithelial membrane. If rats are maintained on a high-fat diet, there is an increase in the amount of intestinal alkaline phosphatase, indicating a role in the transport/processing of the fats (Young et al., 1981). 1

Another hypothesized function for intestinal alkaline phosphatase is protection from bacterial infection, whereby alkaline phosphatase removes phosphate groups from endotoxin, a lipopolysaccharide (lipid-carbohydrate) molecule that makes up the cell wall of some types of bacteria (Poelstra et al., 1997; Kopojos et al. 2003; Verweij et al. 2004). Endotoxin induces an inflammatory response in the host that can be fatal (septic shock), but there is no toxic response when the phosphate groups have been removed from the endotoxin. In this laboratory, we will be using alkaline phosphatase from bovine (cow) intestine. This particular alkaline phosphatase has a molecular weight of 138,000 daltons (a dalton is a unit of mass approximately equal to the mass of a hydrogen atom). It is a dimer (i.e., has two subunits), so the molecular weight of each monomer is 138,000/2 or 69,000 daltons. The enzyme requires four atoms of Zn ++ per dimer for activity. The Zn ++ atoms are said to be cofactors for the enzyme, meaning that they are necessary for the enzyme to be active. An enzyme with its cofactors is called a holoenzyme; without the cofactors, it is an apoenzyme. apoenzyme + cofactors = holoenzyme Typically, apoenzymes are not active, so when we refer to enzymes, we usually mean holoenzymes. The Enzyme-Catalyzed Reaction The reaction we will examine is the removal of phosphate from the molecule p- nitrophenolphosphate. p-nitrophenolphosphate, or pNPP, is not a natural substrate for the enzyme, but it has properties that make it particularly useful to us. The enzyme removes the phosphate and generates free phosphate (P i ) and p-nitrophenol. While the substrate, p- nitrophenolphosphate, is colorless, the product p-nitrophenol is yellow, so we can follow the reaction progress by measuring the generation of yellow color. O P O - O O - 2 0N + H 2 0 OH 2 0N + - O P - O O - p-Nitrophenol Phosphate p-Nitrophenol (yellow) Phosphate O Substrates that change color when acted upon by an enzyme are called chromogenic substrates and these make studying enzymes significantly easier . P-nitrophenol absorbs light at 410 nm, 2

initial velocity or V 0 . (Why is it important to measure initial velocity?) If we plot V 0 vs. substrate concentration [S], we will see the following curve, a Michaelis-Menten curve, shown in Figure 3.2 below. Substrate Concentration [S] Velocity (v o ) V max ½V max K m Figure 3.2 . The Michaelis-Menten curve. This curve describes the relationship between an enzyme (at constant concentration) and the concentration of S, the enzyme's substrate. v o is the initial rate of product formation in an enzyme-catalyzed reaction. See the text on the following page for descriptions of V max , 1/2V max , and K m. As [S] increases, v o eventually becomes independent of [S]. The velocity at which this occurs is called V max , and it is the fastest that the given amount of enzyme can operate. The [S] that yields 1/2 V max is another important kinetic parameter called the Michaelis-Menten constant, designated K m . K m is essential in that it indicates the [S] at which the enzyme is most effective at altering the rate of the reaction . (Make sure that you understand why this is so.) Enzymes are tightly regulated to maintain homeostasis, and one handy mechanism for regulation is having your enzyme activity change based on substrate availability (Is more substrate around? - increase the rate at which the enzyme works to compensate). In fact, it is frequently found that [S] in vivo is near the K m for an enzyme. You can think of this as a cell exploiting the Michaelis-Menten character of an enzyme. Furthermore, K m is important in understanding many other kinetic parameters of enzyme activity that we will not discuss here. K m and V max are characteristics of a reaction that help characterize the enzyme in question. (How would K m and V max change if you increased the amount of enzyme?) To determine K m and V max , we could determine a set of v 0 values at various concentrations of S, make the graph above and read off the values. As you can imagine, this would not be very 4

accurate since obtaining an accurate value for a number that is approached asymptotically is difficult. The equation that describes the above Michaelis-Menten curve is the following: This is called the Michaelis-Menten equation. Two algebraically inclined fellows by the names of Lineweaver and Burke manipulated the Michaelis-Menten equation to yield the following: m max 0 K ] [ ] [ V v + ⋅ = S S Michaelis-Menten Equation. This is called the Michaelis-Menten equation. Two algebraically inclined fellows by the names of Lineweaver and Burke manipulated the Michaelis-Menten equation to yield the following: ] [ V K V 1 v 1 max m max 0 S ⋅ + = Lineweaver-Burke Equation If you plot 1/ v o vs. 1/[S], you get the following Lineweaver-Burke plot or double-reciprocal plot: 1/v 0 1/[S] 1/V max -1/K m Figure 3.3 . Lineweaver-Burke plot. The slope of the line is K m / V max ; the y-intercept is 1/ V max, and, if we extrapolate the line (i.e., set y = 1/v 0 = 0), the x-intercept is -1/ K m . The use of the double reciprocal plot yields much more accurate values for K m and V max than an interpretation of the Michaelis-Menten curve. In this week's lab, we will determine K m and V max for the enzyme alkaline phosphatase. 5

Now, things get a little messy. The units, μM, are in terms of micromoles per liter. That is what the big M means. If we have a concentration of 27 μM, that means we have 27 micromoles per liter or 27 nanomoles/ml (make sure you understand this conversion). Moreover, our reaction mixture is 3.05 ml, and we want the actual total amount of product formed in our tube, not a concentration. So, 27 nmoles/ml x 3.05 ml = 82 nmoles formed in one minute. This then is how much p-nitrophenol was responsible for the yellow color and represents how much p-nitrophenol was generated by the enzyme in one minute. In summary, you'll need to follow three steps to get from your graph of absorbance vs. time to the amount of product formed per minute: 1. Determine the slope of the straight portion of your curve (units are ∆A 410 /min). 2. Convert this slope from ∆A 410 /min to μM/min by using the Beer-Lambert equation. 3. Convert this concentration to the total amount of product formed in the cuvette by considering the total volume. Report your result in terms of nmol/min. 7

Lab 4: Enzyme Kinetics: Characterization of The Enzyme Alkaline Phosphatase The first section below (part A) allows you to practice the assay protocol and get baseline data. An assay is (generally speaking) a test that tells you something about a substance, in this case, the activity of an enzyme. You will use a given amount of enzyme and substrate and measure the rate of product formation in this part. Part B allows you to estimate K m and V max using different amounts of substrates in the enzyme catalyzed reaction. Materials: 1. Spectrophotometer 2. Clean cuvettes 20 ( 13 x 100 mm test tubes) 3. 0.25M Tris HCl pH 8.0 4. 5 mM p-nitrophenol-phosphate (pNPP) in microcentrifuge tube. 5. Parafilm 6. Alkaline phosphatase (AP enzyme) on ice at TA bench Procedure: Time Course of Alkaline Phosphatase Activity to calculate initial velocity. 1. Turn on the spectrophotometer. Allow the instrument to warm up for 15 minutes before you use it. 2. Make sure the machine is in "Absorbance" mode. 3. Set the wavelength to 410 nm. 4. Label two glass tubes as A and B. To tube A, add 3.0 ml of Tris-HCl buffer at pH 8.0. Then add 25 μ l of the 5 mM stock solution of p-nitrophenolphosphate which is the substrate for the reaction. 5. Cover the tube with a piece of Parafilm and invert the tube two times to mix the solution. Insert the cuvette into the spectrophotometer and blank the machine by pressing the red button. (What is the purpose of this step?) 6. ** One student in a group of two will perform the next step, and the other student will set a timer! 7. Add 20 μl of the AP enzyme to the tube while simultaneously marking the time. Quickly mix the solution by inversion and put the tube back in the Spectrophotometer . 8

B. Protocol for the Determination of K m and V max Values: 1. For this experiment, you will partner with a group of two students across from you to complete the assay. Each group is responsible for two substrate concentrations. Decide among yourselves which concentrations your group is analyzing. This week, you must also create your data tables for recording your results for part B. 2. Prepare a set of two tubes for each substrate concentration . Label the two tubes for each concentration. (For example, 2.5A and 2.5 B for 2.5mM pNPP concentration!) 3. Add 3 ml of Tirs-HCl pH 8 buffer to a cuvette/glass tube and 25 μl of the selected pNPP substrate concentration (mM). Cover the tube with a piece of parafilm and mix by inverting. 4. Blank the spectrophotometer at 410 nm with this solution. 5. Add 20 μl of the AP enzyme to the tube while starting a timer at the same time. Quickly mix the tube by inversion and place the tube in the sample compartment of the spectrophotometer. 6. Read the absorbance at 410 nm as 0 sec and then 30 sec intervals for 4 min. 7. Repeat the assay with the second tube for the same concentration. For this experiment, it is important to have duplicates at each substrate concentration; if they are very different, conduct the assay a third time. 8. Then carry out the same procedure with the remaining pNPP concentrations (1.25 mM, 2.5 mM, 5 mM , 7.5 mM, 10mM). Use the data from part A for the 5 mM substrate concentration . Remember to blank the spectrophotometer with each substrate concentration. Write down your absorbances for each pNPP concentration as shown in the example table below. 9. Make sure to get data from the other group at your table so you have data for all the concentrations. 10. For each substrate concentration, plot absorbance versus time and determine the initial velocity. (Again, why is it important to determine the initial velocity?). 10

11. Then calculate the initial velocity as nmoles of p-nitrophenol generated/minute. Time 5 mM pNPP 5 mM pNPP Average A 410 (Make a table for each concentration.) Tube 1-1 Tube 1-2 Average of 1-1 and 1-2 0 min 0.5 min 1 min 1.5 min 2min 2.5 min 3 min 3.5 min 4 min Now you are ready to summarize all those data in just a couple of graphs, which will allow you to look at patterns in the data and determine Km and Vmax. Before graphing, you must first convert your [S] values from the initial concentration in the microfuge tubes to the substrate concentration in your final reaction volume. Make a simple hyperbolic plot of the data; that is, make a graph in which v o (in nmol/min) is plotted as a function of [S]. Your graph should resemble the typical shape of the Michaelis- Menten plot on page 3-3. Don't forget to include the rate for the 5 mM substrate concentration from part A. Then, make a double-reciprocal plot of the data, that is, a graph of 1/v o vs 1/[S] . It should form a straight line, like the Lineweaver-Burke plot in the background information. Fit a line to the points and determine this reaction's K m and V max values. Remember that the slope of the line is K m / V max , and the y-intercept is 1/ V max . Literature Cited Kapojos J., K. Poelstra, T. Borghuis, A. Van den Berg, H. Baelde, P. Klok, and W. Bakker. 2003. Induction of glomerular alkaline phosphatase after challenge with lipopolysaccharide. International Journal of Experimental Pathology 84:135-144 11