Sandbox 156: Difference between revisions

[http://en.wikipedia.org/wiki/Peptidyl_transferase peptidyl transferase] activity<ref name="Day">PMID: 1544895</ref>. However, when CAT III catalyzes the acetylation of chloramphenicol, the antibiotic can no longer bind to the ribosomes and is rendered inactive. The genes for the enzyme are commonly found on the plasmid of the bacteria and have been found in a numerous bacterial species<ref>PMID: 2268277</ref>.

The multifunctional enzyme consists of three identical subunits with three active sites at the subunit interfaces<ref name="Day"/>. A deep, hydrophobic pocket is formed at the interfaces, allowing for binding of the chloramphenicol substrate. Binding of the second substrate, Acetyl-CoA, is accomplished by passing the molecule through a tunnel in the protein to the active site. The active site of CAT III performs acetylations of chloramphenicol via a [http://en.wikipedia.org/wiki/Ternary_complex ternary complex] mechanism and can accommodate the presence of the first intermediates quite well<ref name="Murray">PMID:2015231</ref>.

Both reactions take place in the active site of CAT III, where Acetyl-CoA is tunneled through from the opposing side of the trimer<ref name="Leslie">PMID: 3288984</ref>.

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 resulting [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="Murray"/>.

The general structure of CAT III is dominated by a six stranded antiparallel <scene name='Sandbox_156/Scene_4/4'>β-sheet</scene> and 5 <scene name='Sandbox_156/Scene_4/3'>α-helices</scene> stacked against the ends and face of the protein, forming a structure known as an "open-faced sandwhich"<ref name="Leslie"/>. An extended β-strand forms an extension of the six stranded sheet to a seven stranded sheet that spans the interface of the subunit<ref name="Leslie"/>.  Three identical monomers associate to form the trimeric protein with two <scene name='Sandbox_156/Scene_7/1'>cobalt</scene> ions acting as [http://en.wikipedia.org/wiki/Cofactor_%28biochemistry%29 cofactors]<ref>PMID: 2271709</ref>. 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>.

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>, which acts as a general base<ref name="Murray"/>. Deprotonation is due to the orientation of His-195, which brings the [http://en.wikipedia.org/wiki/Imidazole imidazole ring] of histidine into contact with the chloramphenicol<ref name="Day"/>. Numerous hydrogen bonds are present and, importantly, van der Waals interactions between the [http://en.wikipedia.org/wiki/Benzene benzene ring] of the <scene name='Sandbox_156/Scene_4/1'>Tyr-25</scene> residue and the imidazole ring<ref name="Day"/>. 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 name="Leslie"/>.