Interpretation: The reaction tends to be zero order when the ethanol concentration is above Km. (iii) At [EtOH] = Km, if we solve the Michaelis-Menton Equation, we will deduce that the initial velocity is half-maximal. Thus, we can conclude that with an increase in ethanol substrate concentration, the measured reaction velocity increase to a maximum value Vmax corresponding to point C which is said to be the saturation state where no more ADH is available to react with ethanol. The Km can also be determined by altering the ethanol concentration at half-maximal velocity.
The asymptotic behaviour of Vmax (as it tends to approach infinity) does not allow for accurate calculation of both the rate constants of the Michaelis-Menton equation. The asymptotic approach of rectangular hyperbola makes it difficult to determine their values accurately on the plot. This can be overcome with linear transformations of the Michaelis-Menton equation with simple extrapolation of Vmax and Km from reaction velocities measured at less saturating substrate concentrations. Two such types of linear transformations are the Lineweaver-Burk plot and the Eadie-Hofstee plot (Enzyme Kinetics, 2003).
The Lineweaver-Burk plot is the most common linear form of Michaelis-Menton equation expressed in the following way: 1/v = Km/Vmax. 1/[EtOH] + 1/Vmax (1) Y =m. x + c The Km value can simply be determined from its slope Km/Vmax or from the x-intercept. To determine the x-intercept, put y=0 in equation (1), 0 = Km/Vmax. 1/ [EtOH] + 1/Vmax -1/Vmax = Km/Vmax. [EtOH] Or -[EtOH] = Km Or 1/[EtOH] = -1/Km Thus the x-intercept would be -1/Km. Table 4: Reciprocals of reaction velocities (v) and [EtOH] to determine rate constants of Michaelis-Menton equations using Lineweaver-Burk plot.
1/v (ml. min/umol) 55. 55 33. 33 20. 83 15. 15 12. 82 12. 20 1/[EtOH] (1/mM) 2 1 0. 4 0. 2 0. 1 0. 05 Figure 3: Linear transformation of Michaelis-Menton Equation by Lineweaver-Burk plot. Thus, y-intercept = 11. 034 (put x=0 in line equation to get y-intercept) 1/Vmax = 11. 034; Vmax = 0. 091 umol/mL/min x-intercept = -11. 034/22. 316 (simply put y=0 in line equation) -1/Km = -0. 4944; Km = 2. 02 mM The Eadie-Hofstee plot would be a better choice simply because the Lineweaver-Burk plot has its substrate concentrations crowded at one side of the graph (Birch, 2007).
v = -Km. v/[EtOH] + Vmax (2) Y = m. x + c Thus if we plot reaction velocity against the ratio reaction velocity/ [EtOH], we could expect the slope to be –Km and y-intercept to be Vmax. The Km can also be determined easily from the x-intercept which when deduced from the above equation turns out to be Vmax/Km. This is the Eadie-Hofstee plot with well placed substrate concentration values. Table 5: Values for reaction velocities (v) and v/[EtOH] determined to sketch the Eadie-Hofstee plot.
v(umol/mL/min) 0. 018 0. 03 0. 048 0. 066 0. 078 0. 082 v/[EtOH] (L/min) 0. 036 0. 03 0. 0192 0. 0132 0. 0078 0. 0041 Figure 4: Linear transformation of Michaelis-Menton Equation by Eadie-Hofstee Plot. To determine the Km value for the enzyme, we can simply compare the equation of the line and equation (2) and find that the Km value is 2. 079 (the slope of the graph). Another way would be to determine the x-intercept corresponding to Vmax/Km. The point at which the line touches the x-axis is found to be (0. 044,0). Therefore, Vmax / Km = 0. 044.
Or, Km = Vmax/0. 044 = 0. 092/0. 044 = 2. 090 mM (this value might be even more accurate than the slope). DISCUSSION The role of various kinetic parameters of alcohol dehydrogenase in Saccharomyces cerevisiae was investigated. From the Eadie-Hofstee plot, the velocity at which the enzyme is completely saturated with ethanol (maximal velocity) is 0. 092 umol/mL/min and the Michaelis-Menton constant value closes to 2. 09.
It is worth noting that a low Km value of ADH obtained shows ethanol is a good substrate for ADH and the ADH activity is good. Leskovac et al.(2002) state that a small Km value is associated with an increase in catalytic efficiency of the enzyme. But, the experimentally determined Km does not seem to match with literature value (Online Comprehensive Enzyme Database).
Different Km values for ADH with different metalloenzymes namely Co2+,Cu2+ and Zn2+ are reported and they are found to be comparatively higher at pH 7 (experiment took place at pH 8. 8) and at room temperature (Leskovac, 2003). The experimental results show that the presence of metalloenzymes has a considerable effect on the catalytic activity of ADH in Saccharomyces cerevisiae.
Leskovac et al. (2002) state that the presence of zinc in the tetramer forms stable complexes with inhibitors such as 1,10-phenanthroline and decrease the enzyme efficiency of ADH. An optimum pH and temperature is highly essential for an enzyme to have a high catalytic efficiency. ADH activity is optimum between 8. 6 and 8. 8 and at room temperature. Very high temperature results in high Km due to denaturing of the enzyme (Leskovac, 2003). Thus, it can be safely stated that enzyme kinetics of ADH is affected by changes in pH, temperature, substrate and enzyme concentration.
Increase in enzyme concentration increases reaction velocity and the saturation curve is steeper allowing for an expected decrease in the Km value indicating higher catalytic performance of the enzyme (Leskovac, 2003). Increase in concentration of ethylene glycol will increase the Km for ADH and a decrease in Vmax because ethylene glycol is a poor substrate for ADH (Megarbane et al. 2005). Addition of this dihydric alcohol will permit ADH to break it down into a series of toxic organic acids like glycoaldehyde, oxalic acid, glycolic acid and glyoxylic acid, causing acidosis.
In human beings, this can cause a serious damage in the nervous system and even renal failure (Megarbane et al. 2005). A better method commonly used nowadays is the use of antidote, fomepizole that works as an effective inhibitor that blocks the conversion of ethylene glycol to prevent its metabolism to its toxic products which introduce acidosis (Brent et al. 1999).
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