Experiment 5 Enzyme Kinetics: The Effect of Yeast Alcohol Dehydrogenase and Coenzyme on the Rate of Oxidation of Ethanol.

Results
Raw data:

Effect of Enzyme Concentration | Reagent | 1 | 2 | 3 | 4 | Buffer B | 2.5 ml | 2.4 ml | 2.3 ml | 2.2 ml | NAD+ | 200 μl | 200 μl | 200 μl | 200 μl | Dilute ADH | 200 μl | 300 μl | 400 μl | 500 μl | 6 M Ethanol | 100 μl | 100 μl | 100 μl | 100 μl | Time 0 sec. | 0.022 | 0.026 | 0.069 | 0.073 | Time 5 sec. | 0.028 | 0.038 | 0.110 | 0.114 | Time 10 sec. | 0.038 | 0.051 | 0.141 | 0.150 | Time 15 sec. | 0.048 | 0.064 | 0.176 | 0.187 | Time 20 sec. | 0.058 | 0.078 | 0.206 | 0.222 | Time 25 sec. | 0.068 | 0.091 | 0.239 | 0.259 | Time 30 sec. | 0.078 | 0.104 | 0.270 | 0.291 | Time 35 sec. | 0.088 | 0.115 | 0.301 | 0.325 | Time 40 sec. | 0.099 | 0.131 | 0.331 | 0.358 | Time 45 sec. | 0.109 | 0.145 | 0.362 | 0.391 | Time 50 sec. | 0.119 | 0.158 | 0.392 | 0.424 | Time 55 sec. | 0.130 | 0.167 | 0.420 | 0.455 | Time 60 sec. | 0.140 | 0.179 | | |

Effect of Coenzyme(NAD) Concentration | Reagent | 1 | 2 | 3 | 4 | Buffer B | 2.4 ml | 2.35 ml | 2.3 ml | 2.25 ml | NAD+ | 10 μl | 50 μl | 100 μl | 150 μl | Dilute ADH | 500 μl | 500 μl | 500 μl | 500 μl | 6 M Ethanol | 100 μl | 100 μl | 100 μl | 100 μl | Time 0 sec. | 0.009 | 0.020 | 0.032 | 0.102 | Time 5 sec. | 0.015 | 0.038 | 0.049 | 0.152 | Time 10 sec. | 0.023 | 0.056 | 0.068 | 0.190 | Time 15 sec. | 0.029 | 0.072 | 0.088 | 0.229 | Time 20 sec. | 0.036 | 0.087 | 0.102 | 0.266 | Time 25 sec. | 0.044 | 0.104 | 0.115 | 0.304 | Time 30 sec. | 0.051 | 0.121 | 0.131 | 0.338 | Time 35 sec. | 0.057 | 0.137 | 0.148 | 0.373 | Time 40 sec. | 0.061 | 0.152 | 0.164 | 0.406 | Time 45 sec. | 0.066 | 0.166 | 0.180 | 0.438 | Time 50 sec. | 0.072 | 0.181 | 0.196 | 0.471 | Time 55 sec. | 0.078 | 0.195 | 0.212 | 0.501 | Time 60 sec. | 0.082 | 0.209 | 0.228 | |

Calculation: Effect of Enzyme Concentration | Reagent | 1 | 2 | 3 | 4 | Buffer B | 2.5 ml | 2.4 ml | 2.3 ml | 2.2 ml | NAD+ | 200 μl | 200 μl | 200 μl | 200 μl | Dilute ADH | 200 μl | 300 μl | 400 μl | 500 μl | 6 M Ethanol | 100 μl | 100 μl | 100 μl | 100 μl | △ A/min | 120 | 156 | 382 | 417 | Concentration of enzyme | 1.11 x 10-9 | 1.67 x 10-9 | 2.22 x 10-9 | 2.78 x 10-9 |

The plot of effect of Enzyme Concentration:

This plot shows that with the increasing of enzyme concentration the reaction velocity increase. However, as the enzyme concentration reach a certain amount, the reaction velocity growing slowly.

Effect of Coenzyme(NAD) Concentration | Reagent | 1 | 2 | 3 | 4 | Buffer B | 2.4 ml | 2.35 ml | 2.3 ml | 2.25 ml | NAD+ | 10 μl | 50 μl | 100 μl | 150 μl | Dilute ADH | 500 μl | 500 μl | 500 μl | 500 μl | 6 M Ethanol | 100 μl | 100 μl | 100 μl | 100 μl | △ A/min | 72 | 180 | 192 | 435 | Concentration of coenzyme | 4.98 x 10-5 | 2.5 x 10-4 | 5 x 10-4 | 7.5 x 10-4 |

The plot of Effect of Enzyme Concentration:

This plot shows that with the concentration of coenzyme increase, the reaction velocity increase.

The plot of 1/v vs. 1/[NAD]:

From this plot we can calculate the Km and Vmax, since: y-intercept = 1/Vmax, and x-intercept = -1/Km or slope = Km/Vmax.

For calculation:

Slope = Km/Vmax = 5 x 10-7
Y-intercept = 1/Vmax = 0.003
So: Vmax = 333.3 Km = 1.67 x 10-4

Discussion
The goal of this experiment is to understand the effects of the concentration of enzyme and coenzyme on reaction kinetics by comparing the absorbance at 340 nm.
The reaction for this experiment: RCH2OH + NAD+ RCHO + NADH + H+
When processing the data, researcher found that although the data show with the increasing concentration of enzyme or coenzyme the reaction velocity will increase, the increasing speed are not very organized. Like the second point in the enzyme reaction and the third point in the coenzyme reaction. This shows that there are some errors happen in the experiment.
In the experiment there many factors can have an impact on the accuracy of the data. First, if the reagent did not mix well, the reaction will not be sufficient. On the other hand, if the reagent did not use accurately, they can have an impact on the result as well since the enzyme reaction is sensitive and efficient. Also because the data is combining from several groups and several machines, thus there could be some personal error happen or the different accuracy of machines. When measure the absorbance each time the cuvette should be rinse well, if it did not, the last time reagent can mix with the new sample and the data will not be accurate.
In order to make the data more accurate, people should pay attention on the mixing of reagent. And for each time of assay, the reagent should mix well. In the meantime, before each assay, the cuvette should be washed efficiently, since the reaction is every sensitive and with a little change on concentration the result can be different. Last, the reagent ADH solution is unstable, it better be newly made and keep in ice bath all the time.
However even with the experiment, people find how the concentration change impact the reaction velocity, but the result appear in the experiment can be different from the theoretical situation, since there are some error happen during the experiment. Therefore, with the experiment, it is only can prove that with the increase concentration of enzyme and coenzyme, the reaction velocity will increase.
The experiment prove that the larger the concentration of the enzyme, the reaction will act faster as more enzymes will have more active sites for the substrate to bind to this will speed up the enzymes activity. But enzyme activity is based on substrate concentration if there is a lot of substrate, the enzyme will work its way through at a constant pace, however towards the end the substrate starts to get rarer then the enzyme will have to take more time to find new substrate to act on so the speed will slow down. About the concentrations of coenzyme, they can affect the max or minimum of metabolic fluxes. Therefore with the increasing concentration of coenzyme the reaction velocity will increase as well.
Conclusion
This experiment proves that with the increase concentration of enzyme and coenzyme, the reaction velocity will increase.
Reference
Anonymous, 2014. Mount Saint Vincent University Department of Chemistry, Chemistry 3502 Intermediary Metabolism Laboratory Manual. Winter 2014. 16.