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== Overview of Esterase ==
== Overview of Esterase ==
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The role in the body this would play based on results. Any potential implications of the enzyme if disrupted based on findings and related enzymes.
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This protein belongs to the esterase family and expresses alpha/beta-hydrolase activity. Esterases are broadly defined as hydrolases that split ester, amide, and/or thioester bonds in certain compounds that cause prodrug activation and/or detoxification. The ability of esterases to hydrolyze various drugs make them an important component in drug metabolism<ref>Fukami, T.; Yokoi, T. The Emerging Role of Human Esterases. Drug Metabolism and Pharmacokinetics 2012, 27 (5), 466–477. https://doi.org/10.2133/dmpk.dmpk-12-rv-042.</ref>. This protein's close association with carboxylesterases also indicates that this protein is potentially involved in the hydrolysis of carboxylic ester bonds. Esterases are most commonly located in the skin and liver of mammals, with carboxylesterases being frequently found in the cytosol and endoplasmic reticulum of the skin<ref>Tokudome, Y.; Katayanagi, M.; Hashimoto, F. Esterase Activity and Intracellular Localization in Reconstructed Human Epidermal Cultured Skin Models. Annals of Dermatology 2015, 27 (3), 269. https://doi.org/10.5021/ad.2015.27.3.269.</ref>. Drug metabolism (drug hydrolysis, drug absorption<ref>Williams, F. M. Clinical Significance of Esterases in Man. Clinical pharmacokinetics 1985, 10 (5), 392–403. https://doi.org/10.2165/00003088-198510050-00002.</ref>, etc.) would be inhibited if the function of an esterase is disrupted.
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== Proposed Function ==
== Proposed Function ==
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SwissDock and Chimera allowed for protein-ligand docking studies. Many of the highlighted interactions were ester-containing ligands and others were susceptible to hydrolysis. The hydrolase activity of the protein needed to be tested with a substrate known to work with hydrolases. This substrate is p-nitrophenyl acetate PNP when hydrolyzed produces a yellow colored product. The yellow product will lead to the solution absorbing more at 405nm. <ref>Zhang, S.; Sun, W.; Xu, L.; Zheng, X.; Chu, X.; Tian, J.; Wu, N.; Fan, Y. Identification of the Para-Nitrophenol Catabolic Pathway, and Characterization of Three Enzymes Involved in the Hydroquinone Pathway, in Pseudomonas Sp. 1-7. BMC Microbiology 2012, 12 (1). https://doi.org/10.1186/1471-2180-12-27.
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SwissDock and Chimera allowed for protein-ligand docking studies. Many of the highlighted interactions were ester-containing ligands and others were susceptible to hydrolysis. The hydrolase activity of the protein needed to be tested with a substrate known to work with hydrolases. This substrate is p-nitrophenyl acetate PNPA when hydrolyzed produces p-nitrophenol (PNP), a yellow colored product. The yellow product will lead to the solution absorbing more at 405nm. <ref>Zhang, S.; Sun, W.; Xu, L.; Zheng, X.; Chu, X.; Tian, J.; Wu, N.; Fan, Y. Identification of the Para-Nitrophenol Catabolic Pathway, and Characterization of Three Enzymes Involved in the Hydroquinone Pathway, in Pseudomonas Sp. 1-7. BMC Microbiology 2012, 12 (1). https://doi.org/10.1186/1471-2180-12-27.
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‌</ref>. This allows for the activity of the enzyme to be tracked by measuring the increase in absorbance over time. The isolated protein was introduced to p-nitrophenyl acetate in a buffer with a determined optimal pH of 6. Enzyme activity was measured via a change in absorbance at 405nm over 80 minutes. The data collected was consistent with that of hydrolases in other studies.<ref>Vázquez-Mayorga, E.; Díaz-Sánchez, Á.; Dagda, R.; Domínguez-Solís, C.; Dagda, R.; Coronado-Ramírez, C.; Martínez-Martínez, A. Novel Redox-Dependent Esterase Activity (EC 3.1.1.2) for DJ-1: Implications for Parkinson’s Disease. International Journal of Molecular Sciences 2016, 17 (8), 1346. https://doi.org/10.3390/ijms17081346.
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‌</ref>. This allows for the activity of the enzyme to be tracked by measuring the increase in absorbance over time. The isolated protein was introduced to p-nitrophenyl acetate in a buffer with a determined optimal pH of 6. Multiple trials can be seen in the figures below displaying the affect of pH on the enzyme activity. Enzyme activity was measured via a change in absorbance at 405nm over 30-80 minutes. The data collected was consistent with that of hydrolases in other studies.<ref>Vázquez-Mayorga, E.; Díaz-Sánchez, Á.; Dagda, R.; Domínguez-Solís, C.; Dagda, R.; Coronado-Ramírez, C.; Martínez-Martínez, A. Novel Redox-Dependent Esterase Activity (EC 3.1.1.2) for DJ-1: Implications for Parkinson’s Disease. International Journal of Molecular Sciences 2016, 17 (8), 1346. https://doi.org/10.3390/ijms17081346.
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'''Figure 5: ''' Trial 2 of 4DIU enzymatic activity measured over time via change in absorbance at 405nm in pH 6 buffer
'''Figure 5: ''' Trial 2 of 4DIU enzymatic activity measured over time via change in absorbance at 405nm in pH 6 buffer
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== Structural highlights of 4DIU ==
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== Structural highlights of 4DIU ==
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<ref>PMID:21638687</ref>
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One of the distinctive structural characteristics of this protein is the alpha/beta-hydrolase (ABH) fold. Evidence for this fold consists of its structure (an open β-sheet surrounded by α-helices) and the presence of what is known as the "catalytic triad" (consisting of a nucleophile, an acid, and histidine)<ref>Holmquist, M. Alpha Beta-Hydrolase Fold Enzymes Structures, Functions and Mechanisms. Current Protein and Peptide Science 2000, 1 (2), 209–235. https://doi.org/10.2174/1389203003381405.</ref>. The presence of the active site residues His A 222, Asp A 192, and Ser A 93, as determined by SPRITE, Chimera, etc., confirms the presence of this catalytic triad.
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[[Image:Activesite.jpeg]]
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'''Figure 6''':Catalytic triad of protein 4DIU
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Another distinctive feature of this protein that demonstrates its identity as an esterase is a coil that correlates to the bioinformatic predictions of Chimera, BLAST, Dali, and Sprite.<ref>PMID:21638687</ref>
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Swiss Dock and Chimera predicted that this protein would have binding sites with an affinity to substrates such as acetate, butyrate, phosphate, proline, decanoate, dodecanoate, etc. Wet lab experiments still must be conducted in order to confirm these predictions.
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[[Image:docking.jpg]]
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'''Figure 7''': Docking of cluster 1.2 of proline with protein 4DIU
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Protein 4DIU was calculated to have a molecular weight of 27.28 kDa. This was found by taking 248*110, 248 is the number of amino acids and 110 is the average weight in daltons of an amino acid. This size was confirmed by running an SDS-Page gel of multiple elutions isolated from a column as well as samples from purification steps along the way.
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[[Image:sGel.jpeg]]
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Highlight the data that helped you come to your conclusion here including any relevant figures. Make sure to include potential substrates and binding sites.
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'''Figure 8''': SDS-Page gel confirming size and presence of protein 4DIU
== Conclusions ==
== Conclusions ==
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Overall, the research question was answered and the hypothesis was correct. Protein 4DIU has been identified as belonging to the esterase family and has hydrolytic activity. It appears that the optimal pH for this enzyme is 6 and it is active with p-nitrophenyl acetate. These results are fairly accurate and precise as two trials were run at a pH of 6 and the same trend was seen. Again, from this data, it is believed that protein 4DIU would be active in these parts of the body. In future experiments, it would be beneficial to test the activity at lower pHs such as 3, 4, and 5. Additionally, testing some of the substrates that were modeled to work to see if the bioinformatic data is trustworthy.
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Overall, the research question was answered and the hypothesis was correct. Protein 4DIU has been identified as belonging to the esterase family and has hydrolytic activity. It appears that the optimal pH for this enzyme is 6 and it is active with p-nitrophenyl acetate. These results are fairly accurate and precise as two trials were run at a pH of 6 and the same trend was seen. Again, from this data, it is believed that protein 4DIU would be active in liver and skin cells. In future experiments, it would be beneficial to test the activity at lower pHs such as 3, 4, 5, and 7. This could rule out certain parts of the body if the lower pH causes inactivation. Additionally, testing some of the substrates that were modeled to work to see if the bioinformatic data is trustworthy.
</StructureSection>
</StructureSection>

Current revision

Structural Model of Protein 4DIU

Drag the structure with the mouse to rotate

References

  1. Fukami, T.; Yokoi, T. The Emerging Role of Human Esterases. Drug Metabolism and Pharmacokinetics 2012, 27 (5), 466–477. https://doi.org/10.2133/dmpk.dmpk-12-rv-042.
  2. Tokudome, Y.; Katayanagi, M.; Hashimoto, F. Esterase Activity and Intracellular Localization in Reconstructed Human Epidermal Cultured Skin Models. Annals of Dermatology 2015, 27 (3), 269. https://doi.org/10.5021/ad.2015.27.3.269.
  3. Williams, F. M. Clinical Significance of Esterases in Man. Clinical pharmacokinetics 1985, 10 (5), 392–403. https://doi.org/10.2165/00003088-198510050-00002.
  4. Zhang, S.; Sun, W.; Xu, L.; Zheng, X.; Chu, X.; Tian, J.; Wu, N.; Fan, Y. Identification of the Para-Nitrophenol Catabolic Pathway, and Characterization of Three Enzymes Involved in the Hydroquinone Pathway, in Pseudomonas Sp. 1-7. BMC Microbiology 2012, 12 (1). https://doi.org/10.1186/1471-2180-12-27. ‌
  5. Vázquez-Mayorga, E.; Díaz-Sánchez, Á.; Dagda, R.; Domínguez-Solís, C.; Dagda, R.; Coronado-Ramírez, C.; Martínez-Martínez, A. Novel Redox-Dependent Esterase Activity (EC 3.1.1.2) for DJ-1: Implications for Parkinson’s Disease. International Journal of Molecular Sciences 2016, 17 (8), 1346. https://doi.org/10.3390/ijms17081346. ‌
  6. Holmquist, M. Alpha Beta-Hydrolase Fold Enzymes Structures, Functions and Mechanisms. Current Protein and Peptide Science 2000, 1 (2), 209–235. https://doi.org/10.2174/1389203003381405.
  7. Herraez A. Biomolecules in the computer: Jmol to the rescue. Biochem Mol Biol Educ. 2006 Jul;34(4):255-61. doi: 10.1002/bmb.2006.494034042644. PMID:21638687 doi:10.1002/bmb.2006.494034042644
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