Enzyme Kinetics and Inhibitors - Lab Report Example

Experimental Study of Enzyme Kinetics and Inhibitors Abstract In this study the kinetics of enzyme catalyzed reaction has been investigated. The effect of substrate concentration and inhibitor on the rate of reaction has been studied. The findings are presented in this report. Introduction Enzymes are biomolecules, mostly proteins. These are very important constituent for any living being as they catalyze biochemical reactions. The molecule on which enzyme acts are termed as substrate and the enzyme converts the substrate into product. Enzyme kinetics is concerned with the relationship of enzyme activity and substrate and product concentrations. The investigation of the kinetic properties on an enzyme provides important insight about the reaction mechanism and metabolic properties and function. Therefore, it is very important to investigate the kinetics of an enzyme to understand its role in different biochemical reactions of importance including metabolic reactions. In this study the relationship between initial rates of reaction and the concentration of one of the substrates, acetyl-CoA has been investigated. Effect of an inhibitor of the reaction, propionyl-CoA has also been investigated. Theory The rate of a reaction catalyzed by an enzyme depends upon the concentration of substrates as well as the physical conditions; temperature, pH, ionic strength, the concentration of co-factors etc. Increasing substrate concentrations leads to increased rate of reaction until a maximum is reached. In this state the enzyme is said to be saturated with the substrate. The relationship between initial velocity (vo) and substrate concentration ([S]) is described by a rectangular hyperbole with vmax being the asymptote. The substrate concentration required to achieve a half maximal velocity is a constant termed the Michaelis constant (Km). This is an important parameter to describe the affinity of the enzyme for its substrate. The relationship between velocity and substrate concentration is described by the Michaelis-Menten equation: …………….. (1) Here vo is the velocity observed at substrate concentration [S]. From a series of incubations with varying substrate concentration one can graphically derive the values of Km and vmax by suitably transforming the equation 1. For example, equation (1) can be rewritten as …………… (2) Now, if (y-axis) is plotted against (x-axis) then the intercept will be and the slope will be. In this manner one can graphically calculate the values of Km and vmax. Equipment and Materials A spectrophotometer fitted with a thermostat to control temperature, cuvettes, micropipettes and a strip chart recorder connected to the spectrophotometer. Besides, following materials were used. 1. Mitochondrial suspension 2. 200mM Tris buffer pH 8.0 3. 15mM potassium oxaloacetate 4. 1.5mM acetyl-CoA 5. 3mM dinitrothiobenzoic acid 6. 0.3% (w/v) triton X-1 00 7. 15mM propionyl-CoA Experimental Procedure 1. Spectrometer, water bath and strip chart recorder were switched on and allowed to warm up for 5minutes and in the meantime the required chemicals, consumables and accessories were checked. 2. Sectrophotometer was set at zero and the chart recorder was set at 5% full scale deflection. 3. Polarity of the connection to the strip chart recorder was checked and by interrupting the light path and getting a corresponding positive response from the strip chart recorder. 4. Chemicals in the following table was added to a 3.0 ml cuvette as per indicated volume and concentration. Sl. No. Component Volume Final Concentration 1 Tris buffer 1.5ml 100m M 2 Acetyl-CoA 100µl 50µM 3 Dithionitrobenzoic acid 100µl 100µM 4 Mitochondrial suspension 100µl 10-15 µg protein 5 Triton X-100 100µl 0.01 % (w/V) 6 Water 1 ml 5. The cuvette was placed in the thermostatted cell holder in the spectrophotometer; the lid was closed and after five minute the strip chart recorder was started. The strip chart paper was marked suitably for identification. 6. The lid was opened and 100 µl potassium oxaloacetate solution (final concentration 0.5mM) was added and monitoring of absorbance at 412nm was continued. Absorbance range corresponding to 90% FSD and the chart speed was noted. 7. This experiment was repeated and the initial rate of reaction (nmol.min-1) was calculated assuming a molar extinction coefficient of 13.6 X 103 M-1.cm-1. 8. Steps 3-7 were repeated using 10 µl, 20 µl, 30 µl, 40 µl, 50 µl, 75 µl acetyl-CoA and the remaining volume was made up was adding water. 9. Repeat 8 in the presence of 100 µl propionyl-CoA (final concentration 0.5mM). Calculation of Results Concentration of the acetyl-CoA was calculated as by dividing its volume by the total cuvettes volume (3 ml) and multiplying the resulting value with the concentration of the original acetyl-CoA solution (5 mM). The rate of reaction was obtained by dividing the change in optical density of the solution with time, initial optical density and the molar extinction coefficient. The values, thus calculated are presented in table 1, below: Table 1: Concentration and Corresponding rate of reaction Concentration (nM) 1/S Rate of Reaction (nMmin-1) 1/vo Rate of Reaction (nMmin-1) 1/vo 16.66667 0.06 3.31712E-06 3.01E+05 2.871E-06 348311.1 33.33333 0.03 6.63423E-05 1.51E+04 4.64396E-06 215333.3 83.33333 0.012 7.07014E-05 1.41E+04 1.10065E-05 90855.56 166.6667 0.006 3.5497E-05 2.82E+04 1.27088E-05 78685.71 250 0.004 0.000136202 7.34E+03 1.3342E-05 74951.11 333.3333 0.003 0.000104914 9.53E+03 1.91061E-05 52339.39 The inverse of the reaction rate was plotted against inverse of the substrate concentration. The graph is shown in figure 1, below: Discussion It can be seen from figure 1 that the relationship between 1/vo and 1/[s] is linear in agreement with the Michaelis-Menten equation. There is parallel upward shift in the curve in presence of the inhibitor propionyl-CoA. This is because the inhibitor is a competitive inhibitor. This means that the substrate acetyl-CoA and the inhibitor propionyl-CoA are competing for the same enzyme. Acetyl-CoA reacts with oxaloacetate to form citrate (http://banon.cshl.edu). Therefore, varying the concentration of oxaloacetate will vary the rate of reaction. If this variation is done in a systematic manner and the rate of reaction is investigated then one can calculate the order of the reaction. As citrate is the product of this reaction, therefore, if citrate was present in the reaction, before the reaction started, then the rate of reaction will be slow in agreement with Le-Chatelier’s principle. Conclusion From this investigation it can be concluded that rate of enzyme catalyzed reaction depends on the concentration of the substrate as per Michaelis-Menten equation and an inhibitor retards the rate of the enzyme catalyzed reaction. Reference http://www.biochemj.org/bj/398/0107/3980107.pdf http://en.wikipedia.org/wiki/Enzyme http://banon.cshl.edu/cgi-bin/eventbrowser?DB=gk_current&FOCUS_SPECIES=Arabidopsis%20thaliana&ID=502378& Model data for Practical Class 4 Annexure 1 Enzyme activity in absence of Propionyl-CoA Time(sec.) Optical density at 412nm Volume 5mM acetyl-CoA in incubations 10µl 20µl 50µl 100µl 150µl 200µl 0 0.133 0.133 0.156 0.174 0.149 0.164 10 0.134 0.153 0.181 0.188 0.195 0.203 20 0.136 0.168 0.213 0.233 0.215 0.234 30 0.139 0.178 0.235 0.265 0.249 0.271 40 0.141 0.189 0.259 0.28 0.292 0.307 50 0.147 0.2 0.283 0.322 0.317 0.344 60 0.151 0.21 0.303 0.349 0.344 0.387 70 0.153 0.218 0.324 0.379 0.379 0.425 80 0.155 0.226 0.343 0.405 0.413 0.461 90 0.156 0.229 0.36 0.431 0.451 0.498 100 0.156 0.233 0.377 0.485 0.479 0.535 110 0.156 0.236 0.39 0.509 0.505 0.573 120 0.156 0.237 0.405 0.537 0.538 0.613 130 0.156 0.238 0.414 0.554 0.567 0.648 140 0.156 0.239 0.424 0.578 0.6 0.68 150 0.156 0.239 0.431 0.6 0.629 0.717 160 0.156 0.239 0.438 0.619 0.654 0.75 170 0.156 0.239 0.441 0.637 0.689 0.788 180 0.156 0.239 0.444 0.654 0.713 0.821 Enzyme activity in presence of Propionyl-CoA Time(sec.) Optical density at 412nm Volume 5mM acetyl-CoA in incubations 10µl 20µl 50µl 100µl 150µl 200µl 0 0.461 0.475 0.481 0.486 0.496 0.508 10 0.464 0.48 0.493 0.5 0.511 0.53 20 0.466 0.485 0.503 0.518 0.522 0.559 30 0.469 0.488 0.513 0.529 0.535 0.577 40 0.471 0.492 0.523 0.543 0.558 0.6 50 0.475 0.498 0.533 0.559 0.563 0.629 60 0.477 0.503 0.543 0.571 0.578 0.65 70 0.478 0.507 0.552 0.585 0.592 0.676 80 0.481 0.512 0.563 0.6 0.606 0.7 90 0.483 0.514 0.574 0.613 0.618 0.725 100 0.485 0.519 0.578 0.627 0.633 0.747 110 0.487 0.524 0.587 0.641 0.645 0.77 120 0.49 0.527 0.595 0.653 0.66 0.795 130 0.491 0.531 0.603 0.666 0.673 0.814 140 0.493 0.535 0.61 0.679 0.688 0.837 150 0.495 0.538 0.618 0.693 0.7 0.864 160 0.497 0.54 0.626 0.705 0.712 0.885 170 0.499 0.543 0.633 0.716 0.726 0.905 180 0.5 0.546 0.639 0.727 0.739 0.926 Read More