After completing your analysis of the different bacterial methyltransferases at MethylTranspharmiX, you are off to learn more about inhibition in their Drug Discovery Group. The company is starting a high throughput screen of potential drug targets against ecoDam I, a DNA adenine methyltransferase. This is a bacterially-expressed enzyme that adds a methyl group to adenine in the bacterial genome. Humans do not have this enzyme, so it is an excellent potential target for new antibiotics. Even more exciting, it might also be able to inhibit antibiotic resistant bacteria.(1) After the first round of screening, you and your supervisors have identified several potential targets. Your group went back to the lab to do more detailed enzyme kinetic inhibition analysis. You are going to need to analyze your enzyme kinetic data to see which of these inhibitors is best. Specifically, you need to see which of them inhibits at the lowest concentration, and determine their inhibition mechanism. After that, you will test them against the mammalian cytosine methyltransferase, Dnmt1, and see if it is significantly inhibited, which would limit its use as a therapeutic target. The inhibitors you will focus on is MTX-A. You may refer to it by its final letter for simplicity's sake in your discussion. Using the dataset of MTX-A, be sure to explain what type of inhibition is observed (competitive, noncompetitive, uncompetitive, mixed), and how well it is inhibiting by stating what inhibitor concentration reduces the velocity below 50 at a substrate [S] of 10 nM. Note: The plot is V (reaction velocity) vs. [S] (substrate concentration) for each inhibitor listed. The concentrations listed in the column headers are the Inhibitor Concentrations (in micromolar). All assays were run with the same amount of Dam I enzyme and substrate values. The I=0/ data is the enzyme alone without inhibitor. Table Note: [S] substrate concentration is in nanomolar nM, V is in % of Vmax of Enzyme alone (I=0) = (100). [I] Inhibitor concentration is in micromolar μM, I = 0 is the enzyme alone without inhibitor present. MTX-A: [S] I = 0 I = 0.2 I = 1 I = 5 I = 25 I = 125 0.5 9.1 9.0 8.5 6.59 3.142 0.868 1 16.7 16.2 14.6 9.83 3.727 0.907 2 28.6 27.3 23.1 13.04 4.110 0.928 4 44.4 41.4 32.4 15.58 4.333 0.939 6 54.5 50.0 37.5 16.66 4.412 0.943 10 66.7 60.0 42.9 17.65 4.478 0.946 20 80.0 70.6 48.0 18.46 4.529 0.948 30 85.7 75.0 50.0 18.75 4.546 0.949 50 90.9 78.9 51.7 18.99 4.560 0.950 100 95.2 82.2 53.1 19.17 4.570 0.950 1000 99.5 85.3 54.4 19.33 4.580 0.951 10000 100.0 85.7 54.5 19.35 4.581 0.951
Molecular Techniques
Molecular techniques are methods employed in molecular biology, genetics, biochemistry, and biophysics to manipulate and analyze nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)), protein, and lipids. Techniques in molecular biology are employed to investigate the molecular basis for biological activity. These techniques are used to analyze cellular properties, structures, and chemical reactions, with a focus on how certain molecules regulate cellular reactions and growth.
DNA Fingerprinting and Gel Electrophoresis
The genetic makeup of living organisms is shown by a technique known as DNA fingerprinting. The difference is the satellite region of DNA is shown by this process. Alex Jeffreys has invented the process of DNA fingerprinting in 1985. Any biological samples such as blood, hair, saliva, semen can be used for DNA fingerprinting. DNA fingerprinting is also known as DNA profiling or molecular fingerprinting.
Molecular Markers
A known DNA sequence or gene sequence is present on a chromosome, and it is associated with a specific trait or character. It is mainly used as a genetic marker of the molecular marker. The first genetic map was done in a fruit fly, using genes as the first marker. In two categories, molecular markers are classified, classical marker and a DNA marker. A molecular marker is also known as a genetic marker.
DNA Sequencing
The most important feature of DNA (deoxyribonucleic acid) molecules are nucleotide sequences and the identification of genes and their activities. This the reason why scientists have been working to determine the sequences of pieces of DNA covered under the genomic field. The primary objective of the Human Genome Project was to determine the nucleotide sequence of the entire human nuclear genome. DNA sequencing selectively eliminates the introns leading to only exome sequencing that allows proteins coding.
After completing your analysis of the different bacterial methyltransferases at MethylTranspharmiX, you are off to learn more about inhibition in their Drug Discovery Group. The company is starting a high throughput screen of potential drug targets against ecoDam I, a DNA adenine methyltransferase. This is a bacterially-expressed enzyme that adds a methyl group to adenine in the bacterial genome. Humans do not have this enzyme, so it is an excellent potential target for new antibiotics. Even more exciting, it might also be able to inhibit antibiotic resistant bacteria.(1)
After the first round of screening, you and your supervisors have identified several potential targets. Your group went back to the lab to do more detailed enzyme kinetic inhibition analysis. You are going to need to analyze your enzyme kinetic data to see which of these inhibitors is best. Specifically, you need to see which of them inhibits at the lowest concentration, and determine their inhibition mechanism. After that, you will test them against the mammalian cytosine methyltransferase, Dnmt1, and see if it is significantly inhibited, which would limit its use as a therapeutic target.
The inhibitors you will focus on is MTX-A. You may refer to it by its final letter for simplicity's sake in your discussion.
Using the dataset of MTX-A, be sure to explain what type of inhibition is observed (competitive, noncompetitive, uncompetitive, mixed), and how well it is inhibiting by stating what inhibitor concentration reduces the velocity below 50 at a substrate [S] of 10 nM.
Note: The plot is V (reaction velocity) vs. [S] (substrate concentration) for each inhibitor listed. The concentrations listed in the column headers are the Inhibitor Concentrations (in micromolar). All assays were run with the same amount of Dam I enzyme and substrate values. The I=0/ data is the enzyme alone without inhibitor.
Table Note: [S] substrate concentration is in nanomolar nM, V is in % of Vmax of Enzyme alone (I=0) = (100). [I] Inhibitor concentration is in micromolar μM, I = 0 is the enzyme alone without inhibitor present.
MTX-A:
| [S] | I = 0 | I = 0.2 | I = 1 | I = 5 | I = 25 | I = 125 |
| 0.5 | 9.1 | 9.0 | 8.5 | 6.59 | 3.142 | 0.868 |
| 1 | 16.7 | 16.2 | 14.6 | 9.83 | 3.727 | 0.907 |
| 2 | 28.6 | 27.3 | 23.1 | 13.04 | 4.110 | 0.928 |
| 4 | 44.4 | 41.4 | 32.4 | 15.58 | 4.333 | 0.939 |
| 6 | 54.5 | 50.0 | 37.5 | 16.66 | 4.412 | 0.943 |
| 10 | 66.7 | 60.0 | 42.9 | 17.65 | 4.478 | 0.946 |
| 20 | 80.0 | 70.6 | 48.0 | 18.46 | 4.529 | 0.948 |
| 30 | 85.7 | 75.0 | 50.0 | 18.75 | 4.546 | 0.949 |
| 50 | 90.9 | 78.9 | 51.7 | 18.99 | 4.560 | 0.950 |
| 100 | 95.2 | 82.2 | 53.1 | 19.17 | 4.570 | 0.950 |
| 1000 | 99.5 | 85.3 | 54.4 | 19.33 | 4.580 | 0.951 |
| 10000 | 100.0 | 85.7 | 54.5 | 19.35 | 4.581 | 0.951 |
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