Sandbox Reserved 1052: Difference between revisions
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== '''Introduction''' == | == '''Introduction''' == | ||
[http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis ''Mycobacterium tuberculosis''] is the bacteria that causes the [http://www.mayoclinic.org/diseases-conditions/tuberculosis/basics/definition/con-20021761 tuberculosis] (TB) disease itself, a leading infectious cause of death world-wide. Thus, it is necessary to develop antimycobacterial drugs. However, this has proven to be difficult as the bacteria can become resistant as a result of misuse of drugs. This gives the bacteria time to mutate which ultimately leads to the drug resistance. To combat this, scientists and researchers have taken on many studies and clinical trials to locate specific drug targets. Obstacles for controlling TB infection include lengthy treatment regimens, drug resistance, lack of a highly efficacious vaccine, and incomplete understanding of the factors that control virulence and disease progression.<ref> Online Mendelian Inheritance in Man (OMIM). Mycobacterium tuberculosis, susceptibility to. [Internet]. Baltimore (MD): Johns Hopkins University; 2014 Nov 12 [cited 2015 March 16]. Available from http://www.omim.org/entry/607948 </ref> Of these projects, some deal specifically with the ''M. tuberculosis'' [http://onlinelibrary.wiley.com/doi/10.1046/j.1472-765x.2002.01091.x/epdf Antigen 85] (Ag85) complex. Ag85 complex consists of 3 secreted enzymes (A, B, and C) and plays a key role in pathogenesis and cell wall synthesis of ''M. tuberculosis''. <ref name="Favrot"> PMID:25028518 </ref> | [http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis ''Mycobacterium tuberculosis''] is the bacteria that causes the [http://www.mayoclinic.org/diseases-conditions/tuberculosis/basics/definition/con-20021761 tuberculosis] (TB) disease itself, a leading infectious cause of death world-wide. Thus, it is necessary to develop antimycobacterial drugs. However, this has proven to be difficult as the bacteria can become resistant as a result of misuse of drugs. This gives the bacteria time to mutate which ultimately leads to the drug resistance. To combat this, scientists and researchers have taken on many studies and clinical trials to locate specific drug targets. Obstacles for controlling TB infection include lengthy treatment regimens, drug resistance, lack of a highly efficacious vaccine, and incomplete understanding of the factors that control virulence and disease progression.<ref> Online Mendelian Inheritance in Man (OMIM). Mycobacterium tuberculosis, susceptibility to. [Internet]. Baltimore (MD): Johns Hopkins University; 2014 Nov 12 [cited 2015 March 16]. Available from http://www.omim.org/entry/607948 </ref> Of these projects, some deal specifically with the ''M. tuberculosis'' [http://onlinelibrary.wiley.com/doi/10.1046/j.1472-765x.2002.01091.x/epdf Antigen 85] (Ag85) complex. Ag85 complex consists of 3 secreted enzymes (A, B, and C) and plays a key role in pathogenesis and cell wall synthesis of ''M. tuberculosis''. <ref name="Favrot"> PMID:25028518 </ref> | ||
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== '''Structure''' == | == '''Structure''' == | ||
[[Image:AG85C homodimer.jpg |100 xp|left|thumb|'''Figure 1.''' Ag85C homodimer. ]] | |||
Ag85 C is a <scene name='69/694219/Dimer/1'>dimer</scene> of identical subunits (Figure 1), and quite a bit about the amino acid sequence and overall structure of Ag85C is known. The enzyme contains a [http://en.wikipedia.org/wiki/Catalytic_triad catalytic triad] comprised of [http://en.wikipedia.org/wiki/Serine serine], [http://en.wikipedia.org/wiki/Glutamic_acid glutamate], and [http://en.wikipedia.org/wiki/Histidine histidine]. | Ag85 C is a <scene name='69/694219/Dimer/1'>dimer</scene> of identical subunits (Figure 1), and quite a bit about the amino acid sequence and overall structure of Ag85C is known. The enzyme contains a [http://en.wikipedia.org/wiki/Catalytic_triad catalytic triad] comprised of [http://en.wikipedia.org/wiki/Serine serine], [http://en.wikipedia.org/wiki/Glutamic_acid glutamate], and [http://en.wikipedia.org/wiki/Histidine histidine]. | ||
This would lead us to think that we could inhibit mycolyl transfer and shut down the enzyme by directly inhibiting the serine residue of the <scene name='69/694218/Catalytic_triad/2'>catalytic triad</scene>, but we can instead modify a near by [http://en.wikipedia.org/wiki/Cysteine cysteine] residue and have the same effect. This was determined by a series of experiments in which scientists generated various mutants and modifications of the Ag85C enzyme.<ref> Research Collaboratory for Structural Bioinformatics Protein Database (RCSB PDB) Diacylglycerol acyltransferase/mycolyltransferase Ag85C - P9WQN9 (A85C_MYCTU). [Internet] San Diego (CA): University of San Diego; 2014 [cited 2015 March 16]. [http://dx.doi.org/10.2210/pdbp9wqn9/pdb DOI:10.2210/pdbp9wqn9/pdb]</ref> | This would lead us to think that we could inhibit mycolyl transfer and shut down the enzyme by directly inhibiting the serine residue of the <scene name='69/694218/Catalytic_triad/2'>catalytic triad</scene>, but we can instead modify a near by [http://en.wikipedia.org/wiki/Cysteine cysteine] residue and have the same effect. This was determined by a series of experiments in which scientists generated various mutants and modifications of the Ag85C enzyme.<ref> Research Collaboratory for Structural Bioinformatics Protein Database (RCSB PDB) Diacylglycerol acyltransferase/mycolyltransferase Ag85C - P9WQN9 (A85C_MYCTU). [Internet] San Diego (CA): University of San Diego; 2014 [cited 2015 March 16]. [http://dx.doi.org/10.2210/pdbp9wqn9/pdb DOI:10.2210/pdbp9wqn9/pdb]</ref> | ||
===Active Site=== | ===Active Site=== | ||
[[Image:AG85C active site 3.jpg |100 xp|left|thumb|'''Figure 2.'''Ag85C with labeled active site residues. The three catalytic residues, Ser124, Glu228, and His260, as well as Cys209 are labeled.]] Within the <scene name='69/697503/Active_site/2'>Ag85C active site</scene>,the homodimers shown in blue and green and the active site shown in white, three residues function together to make up the <scene name='69/694219/Cattri/1'>catalytic triad</scene> for this enzyme (Figure 2). The goal of the catalytic triad is to generate a nucleophilic residue for covalent catalysis by using an acid-base-nucleophile triad. These three residues, Ser124, Glu228, and His260 form a charge-relay network to polarize and activate the nucleophile, Ser124, which is then able to attack the substrate to form a covalent intermediate, which is then hydrolysed to regenerate a free enzyme. This charge relay is an example of the well known [http://en.wikipedia.org/wiki/Chymotrypsin chymotrypsin mechanism]. Overall, it is suggested that increased enzymatic activity is attributed to the components of the active site remaining intact so that the serine nucleophile can react to form an intermediary and stabilize the transition state formed during catalysis. In the native structure, the <scene name='69/694219/Helix/2'>α-helix 9</scene> maintains a kinked conformation necessary for correct formation of the hydrogen bonding network between the residues of the catalytic triad, thus allowing for high enzymatic activity.<ref name="Favrot"/> | [[Image:AG85C active site 3.jpg |100 xp|left|thumb|'''Figure 2.''' Ag85C with labeled active site residues. The three catalytic residues, Ser124, Glu228, and His260, as well as Cys209 are labeled.]] Within the <scene name='69/697503/Active_site/2'>Ag85C active site</scene>,the homodimers shown in blue and green and the active site shown in white, three residues function together to make up the <scene name='69/694219/Cattri/1'>catalytic triad</scene> for this enzyme (Figure 2). The goal of the catalytic triad is to generate a nucleophilic residue for covalent catalysis by using an acid-base-nucleophile triad. These three residues, Ser124, Glu228, and His260 form a charge-relay network to polarize and activate the nucleophile, Ser124, which is then able to attack the substrate to form a covalent intermediate, which is then hydrolysed to regenerate a free enzyme. This charge relay is an example of the well known [http://en.wikipedia.org/wiki/Chymotrypsin chymotrypsin mechanism]. Overall, it is suggested that increased enzymatic activity is attributed to the components of the active site remaining intact so that the serine nucleophile can react to form an intermediary and stabilize the transition state formed during catalysis. In the native structure, the <scene name='69/694219/Helix/2'>α-helix 9</scene> maintains a kinked conformation necessary for correct formation of the hydrogen bonding network between the residues of the catalytic triad, thus allowing for high enzymatic activity.<ref name="Favrot"/> | ||
===Cysteine 209=== | ===Cysteine 209=== | ||
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[[Image:Ag85 ebselen.jpg |100 xp|left|thumb|'''Figure 3.''' [http://proteopedia.org/wiki/index.php/4qdu Ag85C-ebselen]: Ebselen covalently binds to Cys209 and forces the otherwise kinked helix α-9 to take on a relaxed conformation, thus allowing movement of the helix and a reduction in enzymatic activity. The native structure is shown in blue while the modified version is shown in purple, demonstrating a relaxed helix. Ebselen-bound Cys209 is shown in green.]] | [[Image:Ag85 ebselen.jpg |100 xp|left|thumb|'''Figure 3.''' [http://proteopedia.org/wiki/index.php/4qdu Ag85C-ebselen]: Ebselen covalently binds to Cys209 and forces the otherwise kinked helix α-9 to take on a relaxed conformation, thus allowing movement of the helix and a reduction in enzymatic activity. The native structure is shown in blue while the modified version is shown in purple, demonstrating a relaxed helix. Ebselen-bound Cys209 is shown in green.]] | ||
[[Image:ebselen pic.jpg |100 xp| | [[Image:ebselen pic.jpg |100 xp|left|thumb|'''Figure 4.''' Ebselen.]] | ||
<scene name='69/694218/Ebselen/1'>Ag85C-Ebselen</scene> (Figure 3) is characterized by a covalent bond between [http://en.wikipedia.org/wiki/Ebselen ebselen](figure 4) and Cys209, thus forcing the otherwise kinked helix α-9 to take on a relaxed conformation (Figure 3). This [http://www.jbc.org/content/289/36/25031/F4.expansion.html alteration of Cys209] allows movement of the helix and causes disruption of the hydrogen bonds within the catalytic triad, ultimately inactivating Ag85C.<ref name="Favrot"/> | <scene name='69/694218/Ebselen/1'>Ag85C-Ebselen</scene> (Figure 3) is characterized by a covalent bond between [http://en.wikipedia.org/wiki/Ebselen ebselen](figure 4) and Cys209, thus forcing the otherwise kinked helix α-9 to take on a relaxed conformation (Figure 3). This [http://www.jbc.org/content/289/36/25031/F4.expansion.html alteration of Cys209] allows movement of the helix and causes disruption of the hydrogen bonds within the catalytic triad, ultimately inactivating Ag85C.<ref name="Favrot"/> | ||
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[[Image:P-chloromercuribenzoic acid.jpg |100 xp| | [[Image:P-chloromercuribenzoic acid.jpg |100 xp|left|thumb|'''Figure 6.''' p-chloromercuribenzoic acid]] | ||