Lab biochemistry - Coursework Example

Determination of the Kinetic constants, Km, Kcat and Vmax of yeast alcohol dehydrogenase (ADH) with three alcohol substrates (ethanol, propan ol and butan-1-ol).IntroductionThe objective of this experiment is to find the Km, kcat and Vmax values of yeast alcohol dehydrogenase. We used ethanol, propan-1-ol and butan-1-ol to carry out this experiment.With a molecular weight of 150.000, yeast dehydrogenase is classified as an NAD linked enzyme. There are four subunits within NAD which consist of Zn2+ ions, the purpose of which is to aid in transfereing a hydride ion from the alcohol to NAD+ .

The overall reaction equation is :RCH2OH + NAD+  RCHO + NADH + H+MethodAs written in the lab protocol sheetResultsTable 1EthanolTimeAbsorbance values for Substrate concentrationsSecondsMinutes0.0800.0400.0200.0100.0050.0000.0000.0000.0000.0000.0000.00010.0000.1670.2670.1200.0810.0730.03520.0000.3330.3690.1650.1260.1330.06130.0000.5000.4390.2640.1690.1780.09840.0000.6670.5500.3180.2170.2160.12750.0000.8330.6000.3840.2580.2520.15560.0001.0000.7250.4390.3080.2870.178Fig.1Fig 1.1Fig 1.2Fig 1.3Fig 1.

4Table 2Propan-1-olTimeAbsorbance values for Substrate concentrationsSecondsMinutes0.080.040.020.010.00500.0000.0000.0000.0000.0000.000100.1670.0180.0410.0360.0330.038200.3330.0530.0610.0440.0390.040300.5000.0850.0850.0620.0520.044400.6670.1150.1110.0790.0350.052500.8330.1430.1370.0970.0760.058601.0000.1680.1590.1120.0870.063701.1670.1960.1840.1280.0990.071Fig 1.5Fig 1.6Fig 1.7 Fig 1.8Fig 1.9Table of absorbance values at 340nm using different substrate concentration for Butan-1-ol and the graphs to show this data.

Table 3Butan-1-olTimeAbsorbance values for Substrate concentrationsSecondsMinutes0.080.040.020.010.00500.0000.0000.0000.0000.0000.000100.1670.0430.030.0290.0270.024200.3330.0780.050.0480.0380.032300.5000.1190.0820.0740.0480.036400.6670.1610.1080.0830.0570.041500.8330.1930.1340.0980.0660.047601.0000.230.160.1160.0760.051701.1670.2640.1830.130.0830.054Fig 2.0Fig 2.1Fig 2.2Fig 2.3Fig 2.4The graphs depicted above have been plotted for for 0.080, 0.040, 0.020, 0.010 and 0.005M substrate concentrations.

All of these graphs have absorbance (340nm) on the y-axis and Time (min) on the x-axis. The graphs were drawn individually however due to a great deal of overlap in the data, it was better to combine it into a single graph for each alcohol as it made the interpretation of the date considerably easier. Michaelis Menten curvesVelocity calculation:1) The linear line equations of the absorbance vs. Time plot. provided the gradient.2) The gradient was divided by the Enzyme co-efficient which in this case was 6220 l/mol/cm3) The above answer was multiplied by the volume of the cuvette which was 0.

003 (3ml÷1000)4) The above answer was multiplied with 106 to convert the volume into micro mol.5) The answer from step 4 in µmol was divided by 0.1 which was the amount of enzyme used.Example for Ethanol:1) 0.851 ÷ 6220 = 1.37 x2) 1.37 x10-4 × 0.003 = 4.10 x10-73) 4.10 x10-7 × 106 = 0.414) 0.41 ÷ 0.1 = 4.1µmol min-1 ml-1The same method was used for the velocity calculation of others. The results have been tabulated above.Ethanol:Table 4EthanolGradientEnzyme co-efficient (l/mol/cm)Cuvette (L)Convert to micromoles (µmol)Enzyme used (µmol min-1 ml0.85162200.00310000000.10.50262200.

00310000000.10.33162200.00310000000.10.35662200.00310000000.10.19262200.00310000000.1Table 5Substrate Concentration (M)Velocity (V)0.0804.1050.0402.4210.0201.5960.0101.7170.0050.926Propan-1-ol:Table 6Propan-1-olGradientEnzyme co-efficient (l/mol/cm )Cuvette (L)Convert to micromolesenzyme used0.17462200.00310000000.10.16562200.00310000000.10.11662200.00310000000.10.09762200.00310000000.10.08062200.00310000000.1Table 7Substrate Concentration (M)Velocity (V)0.0800.8390.0400.7960.0200.5590.0100.4680.0050.386Butan-1-ol:Table 8Butan-1-olGradientEnzyme co-efficient (l/mol/cm )Cuvette (L)Convert to micromolesenzyme used0.23562200.00310000000.10.15962200.

00310000000.10.14462200.00310000000.10.09362200.00310000000.10.06962200.00310000000.1Table 9Substrate Concentration (M)Velocity (V)0.0801.1330.0400.7670.0200.6950.0100.4490.0050.333The Michaelis Menten graphs are attached, fig 2.5, 2.6 and 2.7.Lineweaver Burk plots. Since the velocity had already been calculated from the gradient values of the previous absorbance plots, they could be used in plotting the above mentioned plots. The Graphs of were plotted by dividing the values of velocity and substrate concentrations both by 1 Now that all the info was here, it was possible to plot the Lineweaver Burk plots.

Ethanol:Table 10EthanolSubstrate Concentration (M)Velocity (V)1/S1/V0.0804.10512.5000.2440.0402.42125.0000.4130.0201.59650.0000.6270.0101.717100.0000.5820.0050.926200.0001.080Fig 2.8Propan-1-ol:Table 11Propan-1-olSubstrate Concentration (M)Velocity (V)1/[S]1/V0.0800.00812.500125.0000.0400.00825.000125.0000.0200.00650.000166.6670.0100.005100.000200.0000.0050.004200.000250.000Fig 2.9Butan-1-ol:Table12Butan-1-olSubstrate Concentration (M)Velocity (V)1/S1/V0.0801.13312.5000.8830.0400.76725.0001.3040.0200.69550.0001.4390.0100.449100.0002.2270.0050.333200.0003.003Fig 3.

0Eadie HofsteeThe final set of graphs that needed to be plotted were the Eadie Hofstee plots. These are plots of V/[S] against velocity. All graphs for each alcohol had a negative gradient because of the inverse relationship between the V/S and velocity. Ethanol:Table 13EthanolSubstrate Concentration (M)Velocity (V)V/S0.0804.10551.3130.0402.42160.5250.0201.59679.8000.0101.717171.7000.0050.926185.200Fig 3.1Propan-1-ol:Table 14Propan-1-olSubstrate Concentration (M)Velocity (V)V/S0.0800.83910.4880.0400.79619.9000.0200.55927.9500.0100.46846.8000.0050.38677.200Fig 3.

2Butan-1-ol:Table 15Butan-1-olSubstrate Concentration (M)Velocity (V)V/S0.0801.13314.1630.0400.76719.1750.0200.69534.7500.0100.44944.9000.0050.33366.600Fig 3.3Kcat Calculations1) Step one is: 0.1÷1000= 0.001g/ml2) The molecular weight of the enzyme was 150,000g/mol3) The 0.001 g/ml is divided by 150,000 0.001÷150000= 6.67×mol/ml4) Convert mol to µmol by 6.67×mol/ml × = 6.67µmol/ml5) Kcat= Vmax÷[E]t Kcat = 3.48 ÷ 6.67 = 5217min-11/60 5217 = 86.95 s-1Summary tableVmax, Km and Kcat values for each substrate and each analytical method used have been summarized in the tables below.

Table 16 EthanolPropan-1-olButan-1-olVelocityVmax (V)4.20.851.145Km (M)0.0310.070.015Kcat   Lineweaver BurkVmax (V)3.480.821.076Km (M)0.010.010.011Kcat86.9520.4826.88Eadie HofsteeVmax (V)4.960.9131.25Km (M)0.0260.010.016Kcat123.9322.8131.23Lineweaver BurkFor the Lineweaver Burk method the Vmax and Km values were worked out using the following method:Example for Propan-1-ol:Equation of the linear line: y=0.007x + 1.216Step 1The value 1.216 has been taken from the linear line equation, which in this case is C in the equation.

Step 2Km = Slope VmaxThe value for the slop was taken from the linear line equation which was the gradient. The Vmax in the above equation has already been calculated in step 1.Eadie HofsteeExample for Butan-1-ol:Linear line equation: y=-62.20x + 77.92Y=0To work out the Vmax we want “x” to be the subject therefore:To work out the Km the following equation was used:The -62.20 value was taken from the gradient of the linear line equation. This value of gradient was then by -1 because this value was part of the Eadie Hofstee equation.

DiscussionOnly the first 4 plots were used in making the Absorbance vs. Time because using all the absorbance values would give a curvy orientation to the graph. The reason for this is that the enzyme sites were occupied by the substrates because of which there were no more enzymes sites where the substrate could bind. This is an indication of the Vmax meaning the all the catalytic sites have been occupied and the enzyme has become saturated with the substrate.