The Kinetic Constants of Alkaline Phosphatase were Determined from E. coli K-12 Cells Abstract Alkaline Phosphatase (APase) is an important enzyme in pre-diagnostic treatments making it an intensely studied enzyme. In order to fully understand the biochemical properties of enzymes, a kinetic explanation is essential. The kinetic assessment allows for a mechanism on how the enzyme functions. The experiment performed outlines the kinetic assessment for the purification of APase, which was purified in latter experiments through the lysis of E.coli’s bacterial cell wall. This kinetic experiment exploits the catalytic process of APase; APase catalyzes a hydrolysis reaction to produce an inorganic phosphate and alcohol via an intermediate complex.1 Using the Michaelis-Menton model for kinetic characteristics, the kinetic values of APase were found by evaluating the enzymatic rate using a paranitrophenyl phosphate (PNPP) substrate. This model uses an equation to describe enzymatic rates, by relating the …show more content…
velocity to the substrate concentration1. Using these parameters, the kinetic values KM, Vmax, and kcat were found and calculated. In addition to the Michaelis-Menten plot, three re-arrangements of the Michaelis-Menton equation were also used; Lineweaver-Burke, Eadie-Hofstee, and Hanes-Woolf were plotted and the kinetic values found for these graphs. Comparison of the literature values revealed that the Michaelis-Menton plot gave the most accurate result for KM (0.0176 mM). The the kcat (3.15 sec-1) and turnover rate (1.8 E 5 sec-1 M-1) were then calculated. The Lineweaver-Burke plot illustrated a Km of 0.0106 mM, and a Vmax of 0.0076 μmol/min. The Hanes-Woolf linearization produced a KM of was 0.0092 mML and a Vmax of 0.0074 μmol/min, respectively. The Eadie-Hofstee plot produced a KM of 0.0104 and a Vmax of 0.0076 μmol/min. The most similar literature values found had KM of 0.021 mM, a Kcat of 39.4 sec-1 and a specificity constant of 3.8 E 6. Introduction Enzymes are used to enhance the rate at which reactions occur in organisms. The enzyme of interest in this experiment is Alkaline Phosphatase (APase), which have been isolated from E.coli cells. In order to fully understand enzymatic activity, a kinetic description is required. Generally, the concentration of the substrate [S] varies with the rate of catalysis [Vo]. Given this pathway, the amount of product that is formed will increase until there is a time when equilibrium has been reached. During this time, the enzyme will still be converting substrate into product (and vice versa), but it will have reached its reaction equilibrium. In 1913, Leonor Michaelis and Maud Menton proposed a model that accounts for kinetic characteristics.1 The model proposed: An [E] reacts with [S] to form a [ES] complex with a rate constant of k11.
The [ES] complex can then undergo two different pathways; the complex can dissociate to [E] and [S], at a rate of k or it can shift equilibrium to the left with a rate constant of k2 to form [E] and product [P]1. In this model, the breakdown of the ES complex to yield P is the overall rate-limiting step. Three assumptions of a Michaelis-Menton plot are that a specific [ES] complex in rapid equilibrium between [E] and [S] is a necessary intermediate, the amount of substrate is more than the amount of enzyme so the [S] remains constant, and that this plot follows steady state assumptions. Steady state assumptions states that the intermediate stays the same concentration even if the starting materials and products are constantly changing.2 The rapid equilibrium between enzyme and substrate, and the enzyme-substrate complex yields a mathematical description regarded as the Michaelis-Menton
equation1: At low substrate concentration ([S] < KM), the rate is proportional to the [S] concentration. Vice versa, when the substrate is at high concentrations ([S] < KM), the rate is independent of substrate concentrations. Using this equation, the description of KM can be found. When [S]=KM, the equation will yield: V0=Vmax/2. Therefore, KM is equal to the concentration of the substrate when half of the enzyme’s binding sites are activated. Using the Michealis-Menten pathway, KM can also be written like this1: KM should be run under initial velocity, which is the preliminary linear portion of the Michaelis-Menten plot. The initial velocity is when the initial linear portion of the enzyme reaction has only converted 10% of its product.2 It is then assumed that at this point the substrate concentrations will not change significantly, and the reverse reactions will not affect the rate.2 In order to calculate the substrate concentration correctly, the initial velocity should be used so that the rate of product formation increases with increasing [S] concentrations. If KM is half of Vmax, Vmax must then be when the concentration of the substrate when it is fully saturated. KM is significant, because it can identify the substrate concentration at which catalysis occurs and how tightly the substrate binds to the enzyme. The point at which the substrate concentration is greater than KM is when the rate of catalysis is equal to the turnover rate (kcat). Kcat is the amount of product that has been converted from the substrate in one second. Using kcat, the specificity constant can be calculated. Specificity constant can be used to tell you the catalytic efficiency (turn over rate), which is how fast the enzyme combines with the substrate when the substrate is at Vmax.3 The specificity constant can be used as a purity assessment by comparing the values to known literature values under similar conditions. The kinetic parameters of APase can be found and calculated by plotting the Vi at differing substrate concentrations. The substrate in this instance will be para-nitrophenolphosphate (PNPP) and the product para-nitrophenol (PNP) which is produced by using APase. Once cleaved, the PNP produces a yellow color and the absorbance of this yellow color can be found with a spectrophotometer. Then using Beer’s Law (A=Ɛbc), the rate of the enzyme can be found. Kinetic parameters were determined in the presence of a phosphate acceptor (Tris buffer at pH 8.0), which is a natural inhibitor of APase. Figure one shows an illustrated example of a Michaelis-Menton plot of the reaction velocity (V0) as a function of the substrate concentration [S] for an enzyme that obeys Michaelis-Menten kinetics. At first the catalytic site is empty and awaiting substrate binding. Once the substrate binds, the reaction rate will start to increase linearly with increasing substrate concentrations. The first part of the graph represents a first order reaction, because the rate is dependent on only the substrate concentration. As the substrate concentration increases, the substrate is saturating the enzyme. Once the enzyme has reached maximum saturation, the reaction rate will level off (reach equilibrium). It is during this equilibrium that the reaction is at zero order, meaning that the rate is independent of substrate concentrations. These qualities are what gives the Michaelis-Menton its hyperbolic shape. As shown in the figure, Vmax is approached asymptotically and KM is found during the first linear portion of the graph. The comparison of the kinetic values will assist in the demonstration of the relative purity of the APase sample, as well as give important information about the abilities of this enzyme to perform the intended hydrolysis reaction. Due to the hyperbolic nature of the Michaelis-Menton plot, it is difficult to determine Vmax with precision. There are three common methods for re-arranging the Michaelis-Menton equation to create linear relationships in order to create more precise data points to estimate the Vmax, KM, and kcat values. Lineweaver-Burk (also called the double reciprocal) plots the reciprocal of initial velocity versus the reciprocal of the substrate concentrations. The linear equation for this plot is: Vmax is calculated as the reciprocal of the y-intercept of the best fit line, and KM is obtained by multiplying the slope of the line by Vmax.4 Eadie-Hofstee plots velocity over substrate versus velocity. The linear equation for this plot rearranges to: The intercept is Vmax and the slope is equal to KM.5 In the Hanes-Wolf plot, substrate over velocity is graphed against substrate. The Michaelis-Menton equation is re-arranged to this linear equations: This plot yields the intercept as KM/Vmax and the slope is the reciprocal of Vmax.5 The various methods described give different approximations of these values, due to the assumptions of the distribution of data that each method assumes. A limitation to Lineweaver-Burke is that taking the reciprocal of the velocity gives importance to the smallest values of velocity, which are usually the values that have the greatest percentage error.4 These low values usually signify the area where the lowest amount of product has been formed, therefore it doesn’t give the true values. Eadie-Hofstee and Hanes-Wolf plot fixes the problem of undue importance of velocity on the equation, however there are errors associated with both plots. Eadie Hofstee plot uses velocity as the dependent variable, therefore errors in estimating the reaction rate is amplified leading to errors in the kinetic values. On the other hand, Hanes-wolf plots substrate on both axes, so it is dependent on substrate concentration. Thus, pipetting errors are multiplied which leads to errors associated with the kinetic values. Methods Using a stock solution of 1.0 mM PNPP, a 0.4 mM PNPP solution was made. Stage IV enzyme was then diluted to 65 mU/mL. Measuring Km is an iterative process, therefore this experiment was done with eight different PNPP substrate concentrations (0.1-0.2 mM). 0.1 uL enzyme was in buffer containing 0.2 M Tris was then added to the substrate. All three reagents were then added to a cuvette, ensuring that that the cuvette’s volume was 1 mL. Three trials of the experiment were performed. Using an Evolution 300 Spectrophotometer, the absorbance of PNP was measured for each trial at 400 nm at 30 second intervals for 1 minute. Once the trials were completed, the absorbance values were measured again thirty minutes later to ensure that the reaction had run to completion. A Michaelis-Menton plot, an Eadie-Hofstee plot, a Hanes-Woolf representation, and a Lineweaver-Burk plot were then generated. Using these plots, the Km, Vmax and kcat values were then calculated for each individual plot.
The unknown bacterium that was handed out by the professor labeled “E19” was an irregular and raised shaped bacteria with a smooth texture and it had a white creamy color. The slant growth pattern was filiform and there was a turbid growth in the broth. After all the tests were complete and the results were compared the unknown bacterium was defined as Shigella sonnei. The results that narrowed it down the most were the gram stain, the lactose fermentation test, the citrate utilization test and the indole test. The results for each of the tests performed are listed in Table 1.1 below.
Anne Zhang 3/6/14 BSGE 7-1 Lab Report Problem Paragraph 1 Question: What is the effect of temperature on the dissolving time of an Alka-Seltzer? Alka-Seltzer is made up of baking soda, aspirin, and citric acid which gives the tablet the fizz when dropped in any temperature water. “Alka-Seltzer is a medication that works as a pain reliever and an antacid.
Data table 1 Well plate Contents Glucose concentration A 3 drops 5% sucrose + 3 drops distilled water Negative B 3 drops milk+3 drops distilled water Negative C 3 drops 5% sucrose +3 drops lactase Negative D 3 drops milk +3 drops lactase 15+ E 3 drops 20% glucose +3 drops distilled water 110 ++ Questions B. In this exercise, five reactions were performed. Of those reactions, two were negative controls and one was a positive control.
That familiar fizzing you hear when you drop an Alka Seltzer tablet into a glass of water is the result of a chemical reaction, and chemical reactions are extremely prevalent when it comes to what living things do to carry out life processes. In addition, environmental conditions can alter the results of chemical reactions, and in this lab, we will be answering the
Living organisms undergo chemical reactions with the help of unique proteins known as enzymes. Enzymes significantly assist in these processes by accelerating the rate of reaction in order to maintain life in the organism. Without enzymes, an organism would not be able to survive as long, because its chemical reactions would be too slow to prolong life. The properties and functions of enzymes during chemical reactions can help analyze the activity of the specific enzyme catalase, which can be found in bovine liver and yeast. Our hypothesis regarding enzyme activity is that the aspects of biology and environmental factors contribute to the different enzyme activities between bovine liver and yeast.
One of the most primitive actions known is the consumption of lactose, (milk), from the mother after birth. Mammals have an innate predisposition towards this consumption, as it is their main source of energy. Most mammals lose the ability to digest lactose shortly after their birth. The ability to digest lactose is determined by the presence of an enzyme called lactase, which is found in the lining of the small intestine. An enzyme is a small molecule or group of molecules that act as a catalyst (catalyst being defined as a molecule that binds to the original reactant and lowers the amount of energy needed to break apart the original molecule to obtain energy) in breaking apart the lactose molecule. In mammals, the lactase enzyme is present
The affects of pH, temperature, and salt concentration on the enzyme lactase were all expected to have an effect on enzymatic activity, compared to an untreated 25oC control. The reactions incubated at 37oC were hypothesized to increase the enzymatic activity, because it is normal human body temperature. This hypothesis was supported by the results. The reaction incubated to 60oC was expected to decrease the enzymatic activity, because it is much higher than normal body temperature, however this hypothesis was not supported. When incubated to 0oC, the reaction rate was hypothesized to decrease, and according to the results the hypothesis was supported. Both in low and high pH, the reaction rate was hypothesized to decrease, which was also supported by the results. Lastly, the reaction rate was hypothesized to decrease in a higher salt concentration, which was also supported by the results.
The purpose of this experiment was to discover the specificity of the enzyme lactase to a spec...
Jim Clark. (2007). The effect of changing conditions in enzyme catalysis. Retrieved on March 6, 2001, from http://www.chemguide.co.uk/organicprops/aminoacids/enzymes2.html
That means the active site and the substrate should be exactly complementary so that the substrate can fit in perfectly. Once they collide, the substrate and. some of the side-chains of the enzyme’s amino acids form a temporary. bond so that the substrate can be held in the active site. They combine to form an enzyme-substrate complex and the enzyme can start.
According to the graph on amylase activity at various enzyme concentration (graph 1), the increase of enzyme dilution results in a slower decrease of amylose percentage. Looking at the graph, the amylose percentage decreases at a fast rate with the undiluted enzyme. However, the enzyme dilution with a concentration of 1:3 decreased at a slow rate over time. Additionally, the higher the enzyme dilution, the higher the amylose percentage. For example, in the graph it can be seen that the enzyme dilution with a 1:9 concentration increased over time. However, there is a drastic increase after four minutes, but this is most likely a result of the error that was encountered during the experiment. The undiluted enzyme and the enzyme dilution had a low amylose percentage because there was high enzyme activity. Also, there was an increase in amylose percentage with the enzyme dilution with a 1: 9 concentrations because there was low enzyme activity.
The three-dimensional contour limits the number of substrates that can possibly react to only those substrates that can specifically fit the enzyme surface. Enzymes have an active site, which is the specific indent caused by the amino acid on the surface that fold inwards. The active site only allows a substrate of the exact unique shape to fit; this is where the substance combines to form an enzyme- substrate complex. Forming an enzyme-substrate complex makes it possible for substrate molecules to combine to form a product. In this experiment, the product is maltose.
Various methods such as x-ray crystallography, NMR, and site-directed mutagenesis are applied to study how AP structure contributes to its function and how cofactors and amino acid residues affect reaction mechanism. Enzymes that retain similar structure and function to E. coli AP are found in other species and organisms. For instance, the physiological functions of human AP are still not known at present, but the level of alkaline phosphatase in bloodstream can be a valuable indicator to diagnose liver and bone diseases. Also, mutation in structural gene of human AP will result in hypophosphatasia, a metabolic disease that interfere with uptake of phosphorus and calcium. Thus, understanding the functions of metal ions and reaction mechanism of E. coli AP will provide a general insight of how other enzymes work and discover future potential use of AP in different areas of research or clinical
The experiment was performed using mutated E. coli, cell lysis, two centrifugations, two dialysis processes, heat denaturation, salting out via ammonium sulfate, anion exchange chromatography, and spot testing. By following these procedures, one should be able to obtain a lot of purified alkaline phosphatase and should see a yellow tint during the spot test.
Enzymes are essential biological catalysts in the human body that biochemical reaction. Catalysts work by lowering the activation energy, the minimum energy required for a reaction to occur, which increases the rate of the reaction (Burdge, 2014). Enzymes catalyze reactions by applying pressure onto the bonds of the substrate which lowers the activation energy and breaks these bonds to form products. Even though some enzymes have been found to be non-proteins, most of them are globular proteins which possess an active site where the substrate attaches itself (Raven, 114). The two models that describe the manner in which substrates attach to enzymes are the lock-and-key model and the induced fit model. The lock-and-key model is used to explain an enzyme that fits to only one type of substrate. It is like a lock and key in the sense that only one lock can fit into a key, therefore, only one substrate can fit into the active site of an enzyme that follows this model. On the other hand, an enzyme that follows the induced fit model slightly changes its shape in order for the substrate to...