Determination of the Kinetic Constants With Three Alcohol Substrates - Lab Report Example

Determination of the kinetic constants, Km, Kcat and Vmax of yeast alcohol dehydrogenase(ADH) with three alcohol substrates (ethanol, propan ol andbutan-1-ol Yeast alcohol dehydrogenase (E.C.1.1.1.1) is an NAD-linked enzyme with a molecular weight of 150,000. The enzyme oxidises a number of alcohols, exhibiting classical Michaelis-Menten kinetics, with KM equal to KS, the dissociation constant for the enzyme-alcohol complex. The polypeptide structure of the enzyme protein comprises four identical subunits each containing a Zn2+ ion which facilitates the transfer of a hydride ion from the alcohol to NAD+. The latter must bind to the enzyme before the alcohol; NADH leaves the enzyme after the aldehyde. The overall reaction could be represented as: RCH2OH + NAD+  RCHO + NADH + H+ The reaction is followed spectrophotometrically by measuring the appearance of NADH by its absorbance at 340 nm. The objective of this experiment was to compare the catalytic rate constant, kcat, the Michaelis constant, Km and the apparent second order rate constant, kcat/ Km , for ethanol, propan-1-ol and butan-1-ol. Materials and Methods The various reagents prepared and used in the experiment included the reaction buffer, 0.15 M sodium pyrophosphate with 1mM dithiothreitol, adjusted to pH 8.5 and room temperature; the three substrate solutions, ethanol (0.24M), propan-1-ol (0.24M) and butan-1-ol (0.24M) all adjusted to room temperature; coenzyme NAD+ solution (15mM); yeast alcohol dehydrogenase ( 58 U/mg; Sigma) prepared as a stock solution containing 0.1 mg protein/ml in distilled water and the protein content was accurately determined by measuring the absorbance at 280nm in comparison with a standard solution (1mg/ml) of yeast enolase having A280 value of 0.894. The stock solution of yeast ADH was suitably diluted with 10 mM potassium phosphate buffer, pH 7.4 containing 1 mg/mL of bovine serum albumin to produce A340 /10s of between 0.015 and 0.025 in the ADH assay with ethanol concentration of 80 mM. The absorbance readings were taken using two 3-ml plastic cuvettes in a Novaspec spectrophotometer adjusted to 340nm. The instrument was adjusted to zero absorbance with a cuvette containing 3000 l of distilled water prior to taking the test readings. The assay mixture (total volume, 3000 μl) in the cuvette consisted of 1000μl 0.15 M sodium pyrophosphate buffer with mM dithiothreitol, pH 8; 1000 l of 0.24 M ethanol/propan-1-ol/butan-1-ol (yielding the equivalent of 0.08M ethanol in 3 ml of the assay mixture) or suitable dilutions thereof (yielding 0.04M, 0.02M, 0.01M and 0.005M alcohol substrate, Table 1); 100 l 15 mM NAD+ and the contents mixed well after covering the mouth of the cuvette with parafilm. Lastly, after wiping the optical surfaces of the cuvette, 100 l yeast alcohol dehydrogenase were added and the clock started simultaneously. The solution was mixed well again and the absorbance readings were noted at 10 s intervals over a period of at least one minute. The volume of enzyme added was increased three-fold for butan-1-ol, reducing the volume of water accordingly. Table 1. Showing dilutions of the various alcohol substrates Alcohol (final concentration, M) Alcohol Volume (µL) Water Volume (µL) Ethanol 0.080 1000.0 500.0 Ethanol 0.040 500.0 1000.0 Ethanol 0.020 250.0 1250.0 Ethanol 0.010 125.0 1375.0 Ethanol 0.005 63.0 1437.0 Propanol 0.080 1000.0 500.0 Propanol 0.040 500.0 1000.0 Propanol 0.020 250.0 1250.0 Propanol 0.010 125.0 1375.0 Propanol 0.005 63.0 1437.0 Butanol 0.080 1000.0 500.0 Butanol 0.040 500.0 1000.0 Butanol 0.020 250.0 1250.0 Butanol 0.010 125.0 1375.0 Butanol 0.005 63.0 1437.0 Results and Discussion 1. With ethanol as the substrate. The absorbance readings at 340nm obtained with various concentrations of ethanol are plotted in Figs. 1 to 5. The initial slopes (up to 0.5 min) of the plots were calculated from the regression line. From the values of A340/0.5min obtained, values corresponding to μmol/L/min/ml enzyme NADH formed were calculated thus: (A340 / 6220) x 106 = μmol/L NADH formed in 0.5min. This value when multiplied by 2 gives μmol/L NADH formed per minute. Multiplying the value μmol/L/minute by 10 (since the volume of enzyme used for the reaction was 0.1ml), the value of NADH formed is obtained as μmol/L/min/ml enzyme. The values thus obtained are the initial velocities (V0) at various concentrations of the three alcohol substrates (See Table 2). Fig. 1. Graph showing absorbance values at 340nm obtained with 0.08M ethanol. Slope = 0.628 (determined from A340 values up to 0.5min). Fig.2. Graph showing absorbance values at 340nm obtained with 0.04M ethanol. (Slope = 0.1936, calculated from A340 values up to0.5 min). Fig. 3. Graph showing absorbance values at 340nm obtained with 0.02M ethanol. (Slope = 0.1074, calculated from A340 values up to0.5 min) Fig. 4. Graph showing absorbance values at 340nm obtained with 0.01M ethanol. (Slope = 0.0961, calculated from A340 values up to0.5 min) Fig. 5. Graph showing absorbance values at 340nm obtained with 0.005M ethanol. (Slope = 0.048, calculated from A340 values up to0.5 min). Table 2. Summary of initial rate of reaction with different substrates and their concentrations Substrate alcohol Concentration (μM x 103) V0 (μM NADH/min/ml enzyme) Ethanol 80 2020 Ethanol 40 600 Ethanol 20 340 Ethanol 10 300 Ethanol 5 160 Propan-1-ol 80 200 Propan-1-ol 40 240 Propan-1-ol 20 200 Propan-1-ol 10 140 Propan-1-ol 5 80 Butan-1-ol 80 280 Butan-1-ol 40 60 Butan-1-ol 20 60 Butan-1-ol 10 100 Butan-1-ol 5 20 2. With propan-1-ol as the substrate The absorbance readings at 340nm obtained with various concentrations of propan-1-ol are shown in Figs. 6 to10. The V0 values for 0.08M, 0.04M, 0.02M, 0.01M and 0.005M propoan-1-ol were calculated as described earlier for ethanol and are tabulated in Table 2. Fig.6. Graph showing absorbance values at 340nm obtained with 0.08 M propan-1-ol. (Slope = 0.063, calculated from A340 values up to 0.5 min). Fig. 7. Graph showing absorbance values at 340nm obtained with 0.04 M propan-1-ol. (Slope = 0.073, calculated from A340 values up to 0.5 min). Fig. 8. Graph showing absorbance values at 340nm obtained with 0.02 M propan-1-ol. (Slope = 0.066, calculated from A340 values up to 0.5 min). Fig. 9. Graph showing absorbance values at 340nm obtained with 0.01 M propan-1-ol. (Slope = 0.0405, calculated from A340 values up to 0.5 min). Fig. 10. Graph showing absorbance values at 340nm obtained with 0.005 M propan-1-ol. (Slope = 0.027, calculated from A340 values up to 0.5 min). 3. With butan-1-ol as the substrate The absorbance readings at 340nm obtained with butan-1-ol in the concentration range 0.005M to 0.08M are shown in Figs. 11 to15. The V0 values were calculated for each concentration as described earlier for ethanol and are tabulated in Table 2. Fig. 11. Graph showing absorbance values at 340nm obtained with 0.08 M butan-1-ol. (Slope = 0.0889, calculated from A340 values up to 0.5 min). Fig. 12. Graph showing absorbance values at 340nm obtained with 0.04 M butan-1-ol. (Slope = 0.016, calculated from A340 values up to 0.5 min). Fig.13.Graph showing absorbance values at 340nm obtained with 0.02 M propan-1-ol. (Slope = 0.0209, calculated from A340 values up to 0.5 min). Fig. 14. Graph showing absorbance values at 340nm obtained with 0.01 M butan-1-ol. (Slope = 0.034, calculated from A340 values up to 0.5 min). Fig. 15. Graph showing absorbance values at 340nm obtained with 0.005 M butan-1-ol. (Slope = 0.0065, calculated from A340 values up to 0.5 min). Determination of Km, Kcat and Vmax of yeast alcohol dehydrogenase with ethanol, propan-1-ol and butan-1-ol as substrate To measure the kinetic parameters of an enzyme accurately, the Michaelis-Menten equation needs to be linearised using methods such as the Eadie-Hofstee plot. In this method, V0 is plotted against V0/[S]. The slope of the plot gives (- Km) while the value at the intercept with the Y-axis yields Vmax. Figures 16, 17 and 18 represent Eadie-Hofstee plots for ethanol, propan-1-ol and butan-1-ol as substrates for ADH, respectively. Fig. 16. Eadie-Hofstee plot of yeast ADH using ethanol (5x103μM – 80x103μM) as the substrate. Km = 7181 and Vmax = 855.26 Fig. 17. Eadie-Hofstee plot of yeast ADH using propan-1-ol (5x103μM – 80x103μM) as the substrate. Km = 9386 and Vmax = 263.05 Fig. 18. Eadie-Hofstee plot of yeast ADH using butan-1-ol as the substrate. Km = 3794 and Vmax = 47.693 Fig. 18. Eadie-Hofstee plot of yeast ADH using butan-1-ol (5x103μM – 80x103μM) as the substrate. Km = -9836 and Vmax = 99.67 The turnover number of the enzyme, kcat was calculated for the various substrates from Vmax and enzyme concentration used from the relationship Vmax = kcat /[E] i.e., kcat = Vmax / [E]. Thus, kcat with ethanol was calculated as follows: Vmax = 855.26μmol ethanol /min; Volume of Enzyme used = 0.1ml of 0.1mg enzyme protein /ml or 0.01mg enzyme protein used i.e., 10μg enzyme protein used. In μmoles, therefore, [E] = μg enzyme / molecular weight of enzyme = 10 / 150000 = 6.67 x 10-5 μmoles Substituting for Vmax and [E] and solving the equation for kcat, we get Turnover number kcat = 855.26 / 6.67 x 10-5 = 12822489 min-1 Similarly, kcat propan-1-ol was determined as 263.05 / 6.67 x 10-5 = 3943778 min-1 And, for butan-1-ol, [E] value was multiplied by 3 since the volume of enzyme used was 0.3ml. Therefore, [E] = 2.0 x 10-4 and kcat = 99.67 / 2.0 x 10-4 = 498350 min-1 The results of Km, kcat and kcat/Km for the ethanol, propan-1-ol and butan-1-ol are summarized in Table 3. Table 3. Summary of results of Km, kcat and kcat/Km obtained with ethanol, propan-1-ol and butan-1-ol as substrate for yeast alcohol dehydrogenase Substrate Km kcat kcat / Km Ethanol 7181 12.8 x 106 1785.61 Propan-1-ol 9386 3.9 x 106 420.18 Butan-1-ol -9836 0.5 x 106 -50.66 What do you conclude from a comparison of the kinetic data for the three alcohols? It can be concluded from a comparison of the kinetic data for the three alcohols that yeast ADH has a high affinity for propan -1-ol as suggested by its Km value which is lower than that of ethanol (Table 3). However, the negative value obtained for Km in the case of butan-1-ol suggests that the concentration of substrate might have been too low since the enzyme concentration used had been tripled. The catalytic constant or turnover number, kcat, describes the number of turnovers an enzyme can accomplish with a particular substrate in a given unit of time. Therefore, kcat is a measure of the catalytic efficiency of the enzyme with a particular substrate. As seen from Table 3, the three alcohols differed significantly in their rates of turnover. kcat showed a decreasing trend from ethanol to propan-1-ol to butan-1-ol indicating that the turnover decreases with increasing chain length of the alcohol substrate. While kcat allows a comparison of the relatives velocities of catalysis of an enzyme for different substrates, it does not suggest anything about the affinity of the enzyme for a particular substrate. In contrast, Km is a measure of the affinity, but not of velocity. However, a combination of the two parameters such as the value kcat/Km would be a measure of the efficiency of the catalysis because it indicates the rate of catalysis as a function of the affinity of the enzyme for a substrate. Therefore, higher the kcat/Km value, more efficient the enzyme action. From the values of kcat/Km obtained in this experiment (Table 3), it was evident that the efficiency of yeast alcohol dehydrogenase was the highest with ethanol and followed the order, ethanol > propan-1-ol > butan-1-ol. Again, the negative value of kcat/Km observed with butan-1-ol on account of the negative Km value would suggest zero enzyme activity with this substrate. Read More