Sandbox 156: Difference between revisions

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[[Image:Chloramphenicol.png|thumb|120x150 px|left|Structure of chloramphenicol]]

Chloramphenicol acetyltransferase type III (CAT III) is an enzyme which catalyzes the transfer of the acetyl group from [http://en.wikipedia.org/wiki/Acetyl-CoA ~~acetyl~~-CoA] to hydroxyl groups of [http://en.wikipedia.org/wiki/Chloramphenicol chloramphenicol]. CAT III is a trimeric protein with a ''Mr'' of 25 000-kDa and a member of the [http://en.wikipedia.org/wiki/Acetyltransferase actetyltransferase family] of proteins.

Chloramphenicol acetyltransferase type III (CAT III) is an enzyme which catalyzes the transfer of the acetyl group from [http://en.wikipedia.org/wiki/Acetyl-CoA Acetyl-CoA] to hydroxyl groups of [http://en.wikipedia.org/wiki/Chloramphenicol chloramphenicol]. CAT III is a trimeric protein with a ''Mr'' of 25 000-kDa and a member of the [http://en.wikipedia.org/wiki/Acetyltransferase actetyltransferase family] of proteins.

== Introduction ==

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The multifunctional enzyme consists of three identical subunits with three active sites at the subunit interfaces<ref name="Day"/>. The active site of CAT III performs ~~two~~ acetylations of chloramphenicol and can accommodate the presence of the first intermediates quite well<ref name="Murray">PMID:2015231</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>.

=== Reaction of CAT III ===

------

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>.

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>.

[[Image:Presentation3.jpg|frame|500x450 px|center|Reaction mechanism of CAT III]]

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"/>.

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"/>.

==Structure==

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===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<ref name="Leslie"/>. Instead, the second substrate tunnels to the active site through the

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<ref name="Leslie"/>. 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<ref name="Day"/>. The amino acids lining the tunnel are also primarily hydrophobic producing close contact between the substrate and aromatic residues<ref>PMID:7906544</ref>. This tunneling allows the formation of van der Waals interactions between the [http://en.wikipedia.org/wiki/Adenine 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<ref name="Leslie"/>.

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@@ Line 2: / Line 2: @@
 [[Image:Chloramphenicol.png|thumb|120x150 px|left|Structure of chloramphenicol]]
-Chloramphenicol acetyltransferase type III (CAT III) is an enzyme which catalyzes the transfer of the acetyl group from [http://en.wikipedia.org/wiki/Acetyl-CoA acetyl-CoA] to hydroxyl groups of [http://en.wikipedia.org/wiki/Chloramphenicol chloramphenicol]. CAT III is a trimeric protein with a ''Mr'' of 25 000-kDa and a member of the [http://en.wikipedia.org/wiki/Acetyltransferase actetyltransferase family] of proteins.
+Chloramphenicol acetyltransferase type III (CAT III) is an enzyme which catalyzes the transfer of the acetyl group from [http://en.wikipedia.org/wiki/Acetyl-CoA Acetyl-CoA] to hydroxyl groups of [http://en.wikipedia.org/wiki/Chloramphenicol chloramphenicol]. CAT III is a trimeric protein with a ''Mr'' of 25 000-kDa and a member of the [http://en.wikipedia.org/wiki/Acetyltransferase actetyltransferase family] of proteins.
 == Introduction ==
@@ Line 11: / Line 11: @@
-The multifunctional enzyme consists of three identical subunits with three active sites at the subunit interfaces<ref name="Day"/>. The active site of CAT III performs two acetylations of chloramphenicol and can accommodate the presence of the first intermediates quite well<ref name="Murray">PMID:2015231</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>.
 === Reaction of CAT III ===
 ------
-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>.
+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>.
 [[Image:Presentation3.jpg|frame|500x450 px|center|Reaction mechanism of CAT III]]
-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"/>.
+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"/>.
 ==Structure==
@@ Line 32: / Line 32: @@
 ===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<ref name="Leslie"/>. Instead, the second substrate tunnels to the active site through the
+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<ref name="Leslie"/>. 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<ref name="Day"/>. The amino acids lining the tunnel are also primarily hydrophobic producing close contact between the substrate and aromatic residues<ref>PMID:7906544</ref>. This tunneling allows the formation of van der Waals interactions between the [http://en.wikipedia.org/wiki/Adenine 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<ref name="Leslie"/>.