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Enzymes are important catalysts in the biological system. It is involved in many anabolic and catabolic processes in the body. Enzyme inhibition plays an important role in regulating chemical reactions within the body and therefore, homeostasis. Any changes to these reactions can have a big impact on the human body. Many drugs include enzyme inhibitors to treat illnesses. A prominent example would be the use of the antibiotic penicillin which is shown to inhibit the enzyme transpeptidase and prevent bacterial cell wall formation.
The primary objective of this experiment was to find out if tartronic acid (TA) was an inhibitor of malate dehydrogenase (MDH) and if so, the type of inhibitory effect it had.
We hypothesize that TA, found primarily within cytoplasm of cells and primarily in potatoes outside of cells2, a non-competitive inhibitor of the enzyme MDH.
This investigation made use of the following catalytic oxidation of malate to oxaloacetate using the NAD+/NADH coenzyme system since NADH had a peak absorbance at 340nm which is different from that of NAD+ according to Rost as cited by Held3. Spectrophotometry was utilised to measure changes in absorbance as the reaction progressed.
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Rate of change in absorbance is used to infer reaction rate as NAD+ concentration decreases while NADH concentration increases as reaction progresses. Calculations through the Lineweaver-Burk plot4 and t-test5 was done to conclude that TA was most likely a non-competitive inhibitor of MDH.
The study employed a two-part approach: first, determining L-/D-malate as a suitable substrate for MDH, followed by assessing TA's effect on MDH.
| Reagent | Volume (µl) | Reference Blank | Reagent Blank | L-malate | D-malate |
|---|---|---|---|---|---|
| Type 2 Water | 3000 | 1000 | 1000 | 1000 | 1000 |
| Reaction Buffer | 1750 | 1750 | 1750 | 1750 | 1750 |
| 50mM NAD+ Stock | 150 | 150 | 150 | 150 | 150 |
| 40mM L-malate Stock | - | - | 1000 | - | - |
| 40mM D-malate Stock | - | - | - | 1000 | - |
| MDH Stock | 100 | 100 | 100 | 100 | 100 |
| Total Volume | 3000 | 3000 | 3000 | 3000 | 3000 |
| Reagent | Volume (µl) | With TA | Without TA |
|---|---|---|---|
| Reaction Buffer | 1650 | 1750 | 1750 |
| Malate | 1000 | 1000 | 1000 |
| 50mM NAD+ Stock | 150 | 150 | 150 |
| 20mM Tartronic Acid (TA) | 100 | - | - |
| MDH | 100 | 100 | 100 |
| Total Volume | 3000 | 3000 | 3000 |
Add the MDH enzyme into each cuvette, cover it with parafilm and invert once directly before placing it into the spectrophotometer.
OD readings are taken using the Shimadzu UV1800 spectrophotometer at 340nm across 30s intervals at 0s, 30s, 60s and 90s. at 340nm across 30s intervals at 0s, 30s, 60s and 90s. Using the results obtained, a graph of OD vs time (sec) is plotted to obtain the initial rate of reaction for each substrate. The malate isomer with the higher initial rate is chosen as the suitable substrate for the next part of the investigation.
L-malate was identified as a more reactive substrate for MDH, demonstrating greater increase in absorbance compared to D-malate. The presence of TA significantly decreased the mean Vmax, indicating a non-competitive inhibitory effect on MDH.
| Substrate | Gradient (OD/min) |
|---|---|
| L-malate | 0.0098 |
| D-malate | 0.0003 |
L-malate was chosen for subsequent parts of the investigation due to its higher reaction rate.
| Condition | Vmax (Abs/min) | Km (mM) | P-value |
|---|---|---|---|
| Without TA | 0.10826 | 1.4405 | - |
| With TA | 0.093357 | 1.3317 | - |
| T-test | - | - | 0.012675 (Vmax) |
MDH is added quickly to one cuvette, covered with parafilm and inverted once directly before placing into the spectrophotometer. The spectrophotometer measures OD readings at 10s intervals for 200s. This is repeated for each concentration in cuvettes to obtain the rate of enzymatic reaction as measured by the rate of absorbance for each substrate concentration. To investigate the effect of TA on MDH, 100μl of reaction buffer is replaced with 20mM TA and the experiment is repeated to obtain the reaction rate with the presence of TA.
Each set of absorbance readings are plotted in a graph of change in OD over time/s. The initial rate of each reaction is calculated through the equation of the slope. This is used as the reaction rate, V, for each substrate concentration.
Plot the results obtained in a Lineweaver-Burk plot (1/[V] and 1/[S]) to obtain the Km and Vmax with and without TA present. Average Km and Vmax is calculated for MDH activity without TA (n=27) and with TA (n=14). Since the objective of the experiment is to understand the effect of TA on MDH, a 2-tailed t-test is conducted to test for significant differences between Km and Vmax with and without TA at 5% significance level.
L-malate was a more suitable substrate to measure MDH activity. As graph of OD readings against time was plotted for both L-malate and D-malate, L-malate displayed greater increase in absorbance compared to D-malate. This is shown by the difference in gradient. L-malate trendline had a gradient of 0.0098 while D-malate trendline only had a gradient of 0.0003. L-malate is used when proceeding with the next part of the experiment as it was the more suitable substrate for MDH activity.
According to the t-test conducted, there was a significant decrease in mean Vmax from 0.10826 (SD=0.013060) when TA was absent to 0.93357 (SD=0.022500) when TA was present as the p-value was 0.012675 which was less than 0.05. There was a decrease in mean Km from 1.4405 (SD=0.25178) without TA to 1.3317 (SD=0.46526). However, this change is not significant as p-value was 0.34926 which was greater than 0.05. Therefore, it is most likely that TA was a non-competitive inhibitor of MDH.
Graph of OD readings taken over 90s at 30s intervals for L-malate and D-malate. Both OD readings increased over time. However, The OD readings for L-malate increased at a greater rate with a steeper gradient (gradient = 0.0098). The OD readings for D-malate increased at a much lower rate (gradient = 0.0003).
D-malate and L-malate are 2 compounds with the same chemical formula but different structures. The D- and L- nomenclature is used to differentiate 2 molecules that are mirror image of one another6. D and L isomers are important as they can have different physiological effects 6. The results indicate that L-malate is the more suitable substrate for MDH as it has a higher reaction rate compared to D-malate.
This is corroborated by existing literature which states that most amino acids found in the human body are L-amino acids except glycine which has no chiral center7. MDH is an enzyme that functions in the body, involved in the oxidation of malate to oxaloacetate. Therefore, it is likely that MDH prefer L-malate which is found in the body. L-malate is used to proceed with the experiment and investigate the effect of TA on MDH.
The primary objective of this experiment is to investigate the effect of TA on MDH. Therefore, the t-test is required to compare to the results of the experiment with and without TA present. In the presence of TA, there was a significant decrease in Vmax (p-value0.05) as shown in table above. This is characteristic of non-competitive inhibitors.
According to literature9, non-competitive inhibitors inhibit enzyme activity through binding on allosteric site away from the active site. Non-competitive inhibitors bind to both enzymes and enzyme-substrate complexes differing from uncompetitive inhibitors which only bind to enzyme-substrate complexes. This also differs from the action of competitive inhibitors of competitive inhibitors which bind to active sites of enzymes. This changes the 3D conformation of the active site thus inhibiting the ability of the enzyme to act on its substrate and form products. This results in the decrease in maximum achievable velocity of the reaction and Vmax decreases.
The affinity of enzyme for its substrate remain the same in the presence of non-competitive inhibitors. This is because non-competitive inhibitors do not compete with substrates for the active site on the enzyme. Therefore, the Km value remains the same for non-competitive inhibitors. This contrasts with competitive inhibitors which compete with substrates for the active site. This reduces the affinity of the enzyme for the substrate and Km decreases. Uncompetitive inhibitors result in decrease of Km as well because it reduces the effective concentration of enzyme substrate (ES) complex by forming enzyme substrate inhibitor (ESI) complexes10. This drives the formation of more ES complexes and Km decreases. Since the Km value does not change significantly in the presence of TA, TA is unlikely to be a competitive inhibitor nor an uncompetitive inhibitor.
A significant decrease in Vmax and no significant change in Km of MDH in the presence of TA suggests that TA is in fact a non-competitive inhibitor of MDH.
However, this experiment is limited by the number of replicates. No replicates or repeats were conducted when choosing the suitable substrate for MDH. This could be inaccurate as we are unable to identify any outliers. The experiment conducted with TA present had fewer replicates as well which could affect the overall accuracy of the experiment. More replicates and repeats of the experiment should be conducted to reduce the effect of outliers and increase accuracy.
TA acts as a non-competitive inhibitor of MDH, offering potential therapeutic insights. Further research is recommended to explore TA's broader pharmacological applications.
This experiment taught me the importance of patience and the need to be detail orientated. There were many steps involved in the experiment and some were quite repetitive involving pipetting multiple reagents. However, these steps were very essential and any mistake made could have messed up the whole experiment especially when we were working with such small values. For example, when pipetting the reagents, it is important to slowly pipette reagents to prevent air bubbles from forming as well as to flush out the last drop in the pipettes. If air bubbles form or the last drop is not flushed out, it could change the concentrations of the reagent and impact the results’ accuracy. Despite it being repetitive work, it was important to have patience and always be careful when conducting the experiment.
This practical was also done as group work in groups of six. Hence, some people may not have had the chance to do certain parts of the experiment. However, the work was generally well distributed and I think that each group member contributed well. Everyone was largely on the same page, especially because everyone did their readings.
Additionally, for the part of the experiment where we cover add the enzyme, cover with parafilm and invert, different people did it each time. This could have led to different lag times moving from the table to the spectrophotometer as well as inconsistent mixing. This may have impacted the results of our experiment.
When obtaining the spectrophotometer results, there was not much to do but wait. It made me realise how much time is put into lab work. Just for these few sets of data, we spent around 3 hours in the lab. I can only imagine the amount of time and work researchers invest in the lab.
Our results were not too different from the rest of the class. I think that as a group we tried out best to follow the instructions and be careful to not make any mistakes that could affect the accuracy of our results. I learnt that it is very important to always be very careful and pay attention to all details if you do not want one mistake to impact your results and waste your hard work.
Non-competitive Inhibition of Malate Dehydrogenase by Tartronic Acid. (2024, Feb 16). Retrieved from https://studymoose.com/document/non-competitive-inhibition-of-malate-dehydrogenase-by-tartronic-acid
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