Enzyme Kinetics of Acetylcholinesterase

Enzyme Kinetics of Acetylcholinesterase David Romero Perez Enzyme kinetics of Acetylcholinesterase and its behaviour in the presence of Edrophonium. Abstract The aim of the present study was to test the effects of edrophonium on the enzyme kinetics of acetylcholinesterase. The use of s-acetylthiocholine as a substrate with its breakdown by acetylcholinesterase and the later reaction into a coloured product, allowed the utilization of colorimetric technique in conjunction with spectrophotometry. A Michaelis-Menton and a Lineweaver-Burk plot showed edrophonium to be an acetylcholinesterase inhibitor that does not fit with the classical descriptions of competitive, non-competitive or uncompetitive inhibitors. The results though were coherent with previous research that classed edrophonium as a mixed inhibitor at concentrations similar to the ones used in the present study, 10 uM. On the contrary, the same study suggested that edrophonium behaves as a competitive inhibitor at concentrations of 0.1 uM but this concentration was not tested on the present study and, therefore, further research is required. Introduction Chemical reactions are the fundamental basis of all matter and, therefore, of life. The study of the chemistry relevant to life is called biochemistry and inside this discipline the study of enzymes has been of particular importance. Enzymes simply make rare chemical events common enough to allow the accumulation of, otherwise, improvable molecules or products required for life (Laidler, 1997). Thanks to millions of years of evolution the level of sophistication in biological systems has reached high levels, allowing fine-tuned regulation of enzymes and their products (Berg, Tymoczko and Stryer, 2012). Nonetheless, the study of the enzyme kinetics and how their regulation works had to overcome, with great efforts, the technological difficulties of such small and fast reactions (Laidler, 1997). The first studies done on enzyme kinetics were on fermentation. From ancient cultures to the present humans have use fermentation to produce alcohol and bread. But it was not until the 19th century that fermentation started to be studied. Fischer’s lock and key hypothesis was one of the first successful although not completely accurate attempts to explain the process (Laidler, 1997). On 1902 Brown studied invertase, using yeast and sucrose, discovering the Enzyme-Substrate complex (ES) (Kenneth, 2013). This provided the fundamental blocks for the development of the new-born biochemistry discipline. Another hallmark on biochemistry was the work of Leonor Michaelis and Maud Leonora Menten, 1913, Michaelis-Menten equation (E + S →↠ ES →↠ ES ´ → E + products). Their experiment failed but gave us important lessons on the importance of pH on enzyme reactions (Laidler, 1997). The pH is important because most, if not all, enzymes are active only at specific ranges of pH, and usually reach their optimum activity around 7.0 pH. This value is common in biological systems although specialized enzymes may require higher or lower values (Berg, Tymoczko and Stryer, 2012). Also, the previously mentioned researchers produced an easy way of visualizing the data in the form of a graph called the Michaelis-Menten plot. This graph allows quick recognition of important parameters like the maximum activity reached by the enzyme (Vmax) and the amount of substrate required to produce half Vmax (Km) (Berg, Tymoczko and Stryer, 2012; Laidler, 1997). The Michaelis-Menten plot will be used in this study to show both parameters in relation to the enzyme achetylcholinesterase. Acetylcholinesterase is an enzyme of vital importance for the nervous system. As an enzyme is a globular protein mostly released to the inter-synaptic space between neurons’ axons and dendrites. Its purpose there is to break down the neurotransmitter acetylcholine to prevent it from continuously activating acetylcholine receptors on the post-synaptic neuron (Berg, Tymoczko and Stryer, 2012). As with every enzyme other substances can interact with it or with the conformation of the E+S complex. These components are called inhibitors and are usually described as competitive, non-competitive or uncompetitive, although mixed inhibitors have been also described (Berg, Tymoczko and Stryer, 2012; Howard, 2007). For any chemical to be classed as an inhibitor it must have an negative effect on the Vmax and/or Km. The effect on those would decide what type of inhibitor the chemical is. If competitive the inhibitor binds to the catalytic site and Vmax remains the same while Km is increased. On the other side, if non-competitive, it would bind on a different location than the catalytic site, preventing the binding of the substrate. In this case Vmax would be the same but Km would be decreased. In turn, an uncompetitive inhibitor binds to the Enzyme-Substrate complex (ES) and both Vmax and Km, are decreased (Berg, Tymoczko and Stryer, 2012; Howard, 2007). In the present study the kinetics of achetylcholinesterase are tested in the presence or absence of edrophonium in order to investigate if it is indeed an inhibitor and to which class it belongs. These values were found using a combination of spectrophotometry and colourometry techniques. Spectrophotometry is a technique in which light crosses a cuvette containing the solutes. The content of the solution absorbs a certain amount of light depending on the concentration of the coloured chemical, therefore, less light will reach the detector at the other side of the cuvette. This is called the transmittance, and allows us to calculate the absorbance by subtracting the transmittance to 1 (1-T=A). The absorbance increases or decreases with the capacity of the solution to absorb light, giving an accurate reading of changes in solution composition or concentrations as is the case with enzymes in the presence of their specific substrate (Blauch, 2014; Reed, et al., 1998). This is calculated using the Beer-Lambert law which states that absorbance can be obtained by the equation A=Ecl (E=molar absorbitivity, c=concentration, l=longitude of the path of light which is commonly 1cm) (Anon., n.d.) Being the molar absorptivity (E) of 5-thio-2-nitrobenzoic acid 1.3610^4. The Beer-Lam bert equation can be rearranged (Anon., n.d.) to study the concentrations of unknown samples given that A and E are known and it provides the basis to accurate study of enzyme kinetics together with colourometric technique. Colourometry is based in the natural correlation between the amount of coloured chemical in a solution and the intensity of that colour. Therefore, by comparing solutions of known concentration of the same chemical it is possible to determine the concentration of the unknown concentration sample (Lancashire, 2011). To do so, a spectrophotometer is used by setting it up at the specific wavelength that corresponds to the colour of the reaction (Reed, et al., 1998). In some cases the product of the enzymatic reaction may not produce any colour and a modified substrate can be used. As it was explained before, acetylcholinesterase hydrolyses (breaks down) acetylcholine into an acetyl group and choline. The problem when trying to use the colourometric technique to measure the substrate production is that choline is colourless, hence the reason s-acetylthiocholine is used instead. The break down product thiocholine reacts with 5,5’dithiobis acid (DTNB) to produce 5-thio-2-nitrobenzoic acid (E=1.3610^4). This final product is yellow coloured and can be measured using the spectrophotometer at 412nm wavelength, allowing the precise study of acetylcholinesterase kinetics. Materials The agents used in this experiment were phosphate buffer (0.1 M), acetylthiocholine (15mM), DTNB reagent (6mM), acetylcholinesterase enzyme (0.3 u/ml) and water. All of them provided by UCLan School of Biomedical Sciences. In order to create the mixtures Gilson pipettes ( p20, p200 and p1000) with their respective tips were used. In addition, 3ml tubes were used for the initial adding of agents and 1ml standard plastic cuvettes for the spectrometer, which was also used to measure the absorbance. Methods The present study was divided in three parts. The aim of the first part was to find out the effect of enzyme concentration on rate reaction. The second part aimed to find the effect of different substrate concentration on rate reaction. Finally the third part studied the effect of edrophonium on enzyme rate reaction at different substrate concentrations. As a general note, every single dilution was kept at 3.0ml volume, using phosphate buffer as solvent. Also, every single dilution had 0.1ml AChE but in controls it was replaced with 0.1ml phosphate buffer to keep the 3.0ml volume. All mixtures were produce at room temperature. Plastic cuvettes were used to measure up absorbance in a spectrometer at 412 nm wavelength for two minutes, being the result the average per minute of those two minutes. For the first part of the study on effect of enzyme concentration on rate reaction the mixtures were produced as showed in table 1. AGENT VOLUME 1ST MIXTURE VOLUME 2ND MIXTURE VOLUME 3RD MIXTURE STOCK CONC. REACTION CONC. PHOSPHATE BUFFER 1.25 ml 1.2 ml 1.1 ml 0.1 M 50 mM ACETYLTHIOCHOLINE 0.1 ml 0.1 ml 0.1 ml 15mM 0.5 mM DTNB REAGENT 0.1 ml 0.1 ml 0.1 ml 6 mM 0.2 mM AChE 0.05 ml 0.1 ml 0.2 ml 0.3 u/ml 1st-0.005 u/ml 2nd-0.01 u/ml 3rd-0.02 u/ml WATER 1.5 ml 1.5 ml 1.5 ml n/a n/a Table 1 Reaction Mixtures. Before measuring every mixture the spectrometer was blanked with the correspondent control without the enzyme. The second part of the study looked at the effect on rate reaction of different substrate concentrations. The mixtures were produced with the volumes detailed in table 2. ACETYLTHIOCHOLINE (ml) PHOSPHATE BUFFER (ml) DTNB REAGENT (ml) AChE (ml) WATER Reaction conc of Acetylthiocholine (uM) 0.20 1.1 0.1 0.1 1.5 1000 0.10 1.2 0.1 0.1 1.5 500 0.05 1.25 0.1 0.1 1.5 250 0.02 1.28 0.1 0.1 1.5 100 0.01 1.29 0.1 0.1 1.5 50 0.005 1.295 0.1 0.1 1.5 25 Table 2 Composition of mixtures of acetylcholinesterase enzyme reaction without edrophonium. The effect of edrophonium on rate reaction was studied on the third part of the experiment. The mixtures were produced following table 3. Acetylthiocholine (ml) Phosphate Buffer (ml) DTNB Reagent (ml) Edrophonium (ul) AChE (ml) Water (ml) Reaction conc of acetythiocholine (uM) 0.20 1.1 0.1 100 0.1 1.5 1000 0.10 1.20 0.1 100 0.1 1.5 500 0.05 1.25 0.1 100 0.1 1.5 250 0.02 1.28 0.1 100 0.1 1.5 100 0.01 1.29 0.1 100 0.1 1.5 50 0.005 1.295 0.1 100 0.1 1.5 25 Table 3 Composition of mixtures of acetylcholinesterase enzyme reaction with edrophonium. Once the absorbance was recorded, the Beer-Lambert law equation was transformed to calculate the Velocity of 5-thio-2-nitrobenzoic acid (E=1.3610^4) production in Moles/litre/min achieved by every mixture: -A=ECL → C=A/E (L equals 1 per 1 cm of light path length inside the spectrophotometer cuvettes). The full calculations can be consulted in appendix 1. Results For the first part of the study the effect of enzyme concentration on rate reaction was measured and the velocity on nM/L/min was calculated and noted in table 4. Acetylcholinesterase concentration in u/ml Velocity of reaction in ÃŽ ¼M/L/min 0.005 2.05 0.01 3.97 0.02 7.8 Table 4 Calculated Velocity of reaction by acetylcholinesterase concentration. The velocity was plotted against enzyme concentration in graph 1, which shows a linear relationship between both parameters. Graph 1 Enzyme reaction of acetylcholine in response to enzyme concentration. Next the velocities of enzyme reaction at acetylthiocholine concentrations ranging from 25-1000 ÃŽ ¼M in the presence or absence of edrophonium were calculated and noted in table 5. Reaction concentration of Acetylthiocholine (ÃŽ ¼M) Velocity of reaction without edrophonium (ÃŽ ¼M/L/min) Velocity of reaction with edrophonium (ÃŽ ¼M/L/min) 25 2.5 0.15 50 2.87 0.95 100 3.6 1.25 250 3.75 2.57 500 4.34 2.65 1000 6.62 3 Table 5 calculated Velocities of acetylcholinesterase enzymatic reaction with and without edrophonium. Using the data from table 5 a Michaelis-Menton graph was plotted in graph 2 in order to reveal changes in Vmax and Km in the presence or absence of edrophonium. Graph 2 Michaelis-Menton plot of acetylcholine in the presence or absence of edrophonium. Clear differences on Vmax and Km were found between mixtures with or without edrophonium. In its presence Vmax dropped from 4.34 uM/L/ml to 3.01 uM/L/ml. On the contrary, the amount of substrate (s-acetylthiocholine) required to achieve 50% of Vmax was increased from 30 uM/ml to 100 uM/ml. There was a problem with the higher concentration mixture of the absence condition as it produced a higher than expected absorbance. This was examined in the discussion section. A Lineweaver-Burk plot (graph 3) showed the same results with decreased Vmax and increased Km. Graph 3 Lineweaver-Burk plot acetylcholinesterase in the presence and absence of edrophonium. In agreement with what was observed in graph 2, the graph showed that edrophonium is an acetylcholinesterase inhibitor. The kind of inhibitor it belongs to was examined in the discussion section. Discussion When comparing the Michaelis-Menton and the Lineweaver-Burk plots with the standard results of competitive, non-competitive and uncompetitive inhibitors (Berg, Tymoczko and Stryer, 2012), it became clear edrophonium did not belong to any of those. This can be explained by understanding the mode of action of a given inhibitor with the enzyme-substrate complex. Different inhibitors interact with different parts of a given enzyme or at different moments. A competitive inhibitor â€Å"competes† with the substrate for the catalytic site of the enzyme. As a consequence, the Vmax is reduced but if the concentration of the substrate is increased, more substrate would reach the catalytic site, nullifying the effect of the inhibitor although increasing the Km. An uncompetitive inhibitor does not bind to the catalytic site but somewhere else on the enzyme. It binds only once the E+S complex has been formed, decreasing the reaction rate regardless the substrate concentration. As a result the enzyme can not reach its normal Vmax and the Km is decreased. On the other hand, a noncompetitive inhibitor does not need the E+S complex to bind to the enzyme and does not decrease E+S formation. However, the E+S+I complex would not create a product, inactivating the enzyme. Basically, the noncompetitive inhibitor has taken a percentage of the active enzy me from the population, decreasing the Vmax but maintaining the same Km for the rest of the active enzyme population (Berg, Tymoczko and Stryer, 2012). The results of the present study suggest that edrophonium decreases the Vmax whilst increasing the Km and this effect can not be overcome by increasing substrate concentration. As a result, it can be classed as a mixed inhibitor, which inhibits the binding of the enzyme to the substrate and, at the same time, inactivates a proportion of the enzyme population (Berg, Tymoczko and Stryer, 2012). This has been supported by previous research (Robaire Kato, 1975) that found edrophonium to be a competitive inhibitor at concentrations of 0.1 uM but a mixed inhibitor at concentrations like the used in the present study, 10 uM. There were some limitations with the materials used. Plastic cuvettes were used instead of glass ones which are more suitable for organic solvents (Reed, et al., 1998). Also, the relative pipetting inexperience of the researches might have affected the accuracy of the resulting mixtures, hence the odd results for the mixture of higher substrate concentration on the absence condition. In future research it is recommended to improve pipetting accuracy maybe by using an automated pipetting system. Also, the timing in enzymatic reactions is critical, as these reactions occur often in seconds or even milliseconds (Laidler, 1997). Therefore, a multiplate spectrophotometer reader could be used to measure the absorbance of the mixtures. This would avoid any potential differences and delays from the moment the mixture is done to its reading. Also, lower concentrations of edrophonium (0.1 uM) should be tested to corroborate Robaire and kato’s (1975) research. In conclusion, in agreement with previous research (Bonaire Kato, 1975), the data points at edrophonium as an acetylcholinesterase mixed inhibitor at least at high concentrations (10 uM). Nonetheless, it needs to be confirmed in future research that edrophonium is also a competitive inhibitor at low concentration. At the same time, the technique could be optimized by the use of automated means in order to improve accuracy given the odd results produced by poor pipetting accuracy. References Anon (n.d.) Beers Law. Available: http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm. Last accessed 15th Jan 2014. Berg, J. M., Tymoczko, J. L. and Stryer, L. (2012) Biochemistry, 7th ed. New York: Freeman. Blauch D. N. (2014) Spectrophotomery. Available: http://www.chm.davidson.edu/vce/spectrophotometry/Spectrophotometry.html. Last accessed 15th Jan 2014. Howard, A. J. (2007) Enzyme inhibition and regulation, CSRRi,iit, [online]. Available at: http://csrri.iit.edu/~howard/biochem/lectures/enzymeinhibition.html. Last accessed 15th Jan 2014. Kenneth, A. J. (2013) A century of enzyme kinetic analysis, 1913 to 2013. FEBSLetters. 587, 2753-2766. Laidler, K. J. (1997) A brief history of enzyme kinetics. In: A. Cornish-Bowden ed. New Beer in an Old Bottle: Eduard Buchner and the Growth of Biochemical Knowledge. Valencia: Universitat de Valencia, pp. 127-133. Lancashire, R. J. (2011) EXPERIMENT 36 COLOURIMETRIC DETERMINATION OF PHOSPHATE. Available: http://wwwchem.uwimona.edu.jm/lab_manuals/c10expt36.html. Last accessed 15th Jan 2014. Reed, R. Holmes, D. Weyers, J. Jones, A. (1998) Practical Skills in Biomolecular Sciences. 4th ed. Essex: Pearson. 310-313. Robaire, B., Kato, G. (1975) Effects of Edrophonium, Eserine, Decamethonium, d-Tubocurarine, and Gallamine on the Kinetics of Membrane-Bound and Solubilized Eel Acetylcholinesterase. MOLECULAR PHARMACOLOGY. 11 (6), 722-734. Appendix 1 Velocity calculations Normal absorbances (nM) Divided by E Velociy (ÃŽ ¼M/L/min) 1/Velocity 0.034 2.5 0.4 0.039 2.87 0.35 0.049 1.3610^4 3.6 0.277 0.051 3.75 0.266 0.059 4.34 0.23 0.090 6.62 0.15 absorbances in the presence of edrophonium (nM) Divided by E Velociy (ÃŽ ¼M/L/min) 1/Velocity 0.002 0.15 6.6 0.013 0.95 1.05 0.017 1.3610^4 1.25 0.8 0.035 2.57 0.39 0.036 2.65 0.37 0.041 3 0.33