Sandbox 156: Difference between revisions
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The multifunctional enzyme consists of three identical subunits with three active sites at the subunit interfaces. The side chains of one subunit allow [http://en.wikipedia.org/wiki/Van_der_Waals_force van der Waals interactions] and two hydrogen bonds with chloramphenicol, causing binding of the substrate. The opposing subunit provides a histidine (His-195) residue essential for catalysis<ref>PMID: 8407936</ref>. Water molecules in the cavity provide a bridging hydrogen bond between the 1-hydroxyl of chloramphenicol and the hydroxyl of a threonine (Thr-174) residue. The active site of CAT III performs two acetylations of chloramphenicol and can accommodate the presence of the first intermediates quite well<ref name= | The multifunctional enzyme consists of three identical subunits with three active sites at the subunit interfaces. The side chains of one subunit allow [http://en.wikipedia.org/wiki/Van_der_Waals_force van der Waals interactions] and two hydrogen bonds with chloramphenicol, causing binding of the substrate. The opposing subunit provides a histidine (His-195) residue essential for catalysis<ref>PMID: 8407936</ref>. Water molecules in the cavity provide a bridging hydrogen bond between the 1-hydroxyl of chloramphenicol and the hydroxyl of a threonine (Thr-174) residue. The active site of CAT III performs two acetylations of chloramphenicol and can accommodate the presence of the first intermediates quite well<ref name=”4”> PMID:2015231 </ref>. | ||
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[[Image:Picture2.jpg|500x400 px|center]] | [[Image:Picture2.jpg|500x400 px|center]] | ||
In the first step of the reaction, Histidine-195 abstracts a proton from the 3-hydroxyl of chloramphenicol, promoting a [http://en.wikipedia.org/wiki/Nucleophilic_attack nucleophilic attack] from the [http://en.wikipedia.org/wiki/Oxyanion oxyanion] to the thioester bond of the acetyl-CoA. The intermediate produced, 3-acetylchloramphenicol, then rearranges non-enzymatically to 1-acetylchloramphenicol. Regeneration of the 3-hydroxyl allows another round of CAT III catalyzed nucleophilic attack and a 1,3-diacetylchloramphenicol product is formed<ref name= | In the first step of the reaction, Histidine-195 abstracts a proton from the 3-hydroxyl of chloramphenicol, promoting a [http://en.wikipedia.org/wiki/Nucleophilic_attack nucleophilic attack] from the [http://en.wikipedia.org/wiki/Oxyanion oxyanion] to the thioester bond of the acetyl-CoA. The intermediate produced, 3-acetylchloramphenicol, then rearranges non-enzymatically to 1-acetylchloramphenicol. Regeneration of the 3-hydroxyl allows another round of CAT III catalyzed nucleophilic attack and a 1,3-diacetylchloramphenicol product is formed<ref name=”4”/>PMID:2015231</ref>. | ||
==Structure== | ==Structure== | ||
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===The Chloramphenicol Binding Site=== | ===The Chloramphenicol Binding Site=== | ||
The association of the monomeric subunits produces a well-defined pocket at the subunit interface, allowing for the binding of the chloramphenicol molecule. The <scene name='Sandbox_156/Scene_3/1'>chloramphenicol binding site</scene> of CAT III is lined with hydrophobic residues, allowing | The association of the monomeric subunits produces a well-defined pocket at the subunit interface, allowing for the binding of the chloramphenicol molecule. The <scene name='Sandbox_156/Scene_3/1'>chloramphenicol binding site</scene> of CAT III is lined with hydrophobic residues, allowing only 2 hydrogen bonds with the substrate. A third hydrogen bond is mediated through a <scene name='Sandbox_156/Scene_4/2'>bridging water molecule</scene> and allows interaction between the the 1-hydroxyl of chloramphenicol and the hydroxyl of Tyr-174<ref>PMID:2015231</ref>. | ||
===The Acetyl-CoA Binding Site=== | |||
The binding of chloramphenicol blocks acetyl-CoA from entering the active site from the <scene name='Sandbox_156/Scene9/1'>top of the enzyme</scene>, as illustrated. Instead, the second substrate tunnels to the active site through the | |||
<scene name='Sandbox_156/Scene9/2'>pantetheine arm</scene> from the opposite side of the trimer. The amino acids lining the tunnel are also primarily lined with hydrophobic residues allowing for close contact between the substrate and aromatic residues<ref>PMID: 1544895</ref>. This tunneling allows the formation of van der Waals interactions between the adenine ring of acetyl-CoA with dimethyl groups of the pantetheine arm and hydrogen bonding of the ring with Tyr-56,Phe-96 and Phe-103, binding the substrate. | |||
===The Active Site=== | |||
The active site catalyzes sequential acetylations of the chloramphenicol molecule. This is initiated by the deprotonation of the 3-hydroxyl of chloramphenicol by <scene name='Sandbox_156/Scene_two/1'>His-195</scene><ref>PMID:2015231</ref>. Numerous hydrogen bonds are present and, importantly, van der Waals interactions between the benzene ring of the <scene name='Sandbox_156/Scene_4/1'>Tyr-25</scene> residue and the imidazole ring. This van der Waals contact may help stabilize side-chain orientations, promoting specificity in the reaction. The acetyl group is already properly positioned after tunneling, and reacts readily without any major structural changes<ref>PMID: 3288984</ref>. | |||
Structure used in the above scenes: 3cla | |||
==References== | |||
<references/> | |||
<table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr> | |||
