Sandbox 212: Difference between revisions

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Ndeye Coumba, Alina Spielmann

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 * '''The role of carnitine acyltransferases in fatty acid oxidation'''
 The most important biological function of carnitine acyltransferases is the transport of fatty acids for β-oxidation<ref name="structure" />.Fatty acids are oxidized for energy production in the mitochondrial matrix by a process called β- oxidation. The major site of fatty acid accumulation, however, is the cytoplasm of the cells. Hence, in order to provide energy, fatty acids have to be transported from the cytoplasm across the inner mitochondrial membrane into the mitochondrial matrix. The carnitine shuttle, a transport chain that consists of three enzymatic reactions, helps fatty acids to pass the mitochondrial membrane. Carnitine acyltransferases (CrATs, COTs, CPTs) are part of the carnitine shuttle.
-[[Image:Second step carnitine shuttle.jpg
+[[Image:Second step carnitine shuttle.jpg|thumb|second step of the carnitine shuttle|600px|left]]
-|thumb|second step of the carnitine shuttle|600px|left]]
 The first step of the carnitine shuttle is the activation of fatty acids. They are transformed into the activated form (= acyl CoA) by the formation of a thioester linkage between the fatty acid carboxyl group and the thiol group of coenzyme A. The '''second step''' of the carnitine shuttle is catalyzed by <font color='#802828'>'''carnitine acyltransferases'''</font>. These enzymes (especially CPT) facilitate the transport of fatty acids by conjugating them to <font color='#267DAB'>'''carnitine'''</font>. In this reaction an <font color='#c88033'>'''acyl group'''</font> is transferred from the sulfur atom of <font color='#00007C'>'''CoA'''</font>  to the hydroxyl group of carnitine. The product is <font color='#c88033'>'''acyl-'''</font> <font color='#267DAB'>'''carnitine'''</font>. The third step of the carnitine shuttle is also catalyzed by <font color='#802828'>'''carnitine acyltransferases'''</font> (especially CPT) in the mitochondrial matrix. In this reaction the <font color='#c88033'>'''acyl group'''</font> of <font color='#c88033'>'''acyl-'''</font> <font color='#267DAB'>'''carnitine'''</font> ester is transferred back to <font color='#00007C'>'''CoA'''</font> to form <font color='#c88033'>'''acyl-'''</font> <font color='#00007C'>'''CoA'''</font>. <ref> Lehninger Principles of Biochemistry |ISBN-10: 071677108X | ISBN-13: 978-0716771081 | Publication Date: February 1, 2008 | Edition: 5th </ref>
 == Structure of Carnitine acetyltransferase  ==
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 <StructureSection load='1ndf' size='500' side='right' caption='Structure of carnitine acetyltransferase in complex with carnitine (PDB entry [[1ndf]])' scene='Sandbox_212/Carnitine_acyltransferase/1'>
 * '''overall structure'''
-The structure of CrAT contains 16 β strands (β1–β16) and 20 α helices (α1–α20) and can be divided into two domains, a C domain and a N domain. The shown 3D structure shows two molecules of carnitine acetyltransferases und thus two active sites as well as two carnitine binding sites.
+The structure of CrAT contains 16 β strands (β1–β16) and 20 α helices (α1–α20) and can be divided into two domains, a C domain and a N domain.<ref name="structure1"/> The shown 3D structure shows two molecules of carnitine acetyltransferases and thus two active sites as well as two carnitine binding sites.
 The core of the 2 domains is a 6-stranded mixed β-sheet (β10-β13, β15, β16 in the C domain), with 3 α-helices covering one of its faces (α6, α7, α12 in the N domain). The other face of the β-sheet in the N domain is covered by additional helices, whereas the other face of the β-sheet in the C domain is covered by the N domain.
 * '''active site'''
-The <scene name='Sandbox_212/Active_site/1'>active site</scene> of CrAT is located at the interface between the N and C domains. Biochemical and mutagenesis studies have permitted to identify a histidine residue as the catalytic base of carnitine acyltransferases. It can be reached from two openings of the tunnel on the surface of the structure. One of these openings is used for binding carnitine, while the other is used for binding CoA. Hence, the two substrates of the enzyme bind at opposite sides of the <scene name='Sandbox_212/Histidine343/1'> catalytic  residue</scene>.
+The <scene name='Sandbox_212/Active_site/1'>active site</scene> of CrAT is located at the interface between the N and C domains. Biochemical and mutagenesis studies have permitted to identify a <scene name='Sandbox_212/Histidine343/1'> histidine residue as the catalytic base </scene> of carnitine acyltransferases. It can be reached from two openings of the tunnel on the surface of the structure. One of these openings is used for binding carnitine, while the other is used for binding CoA. Hence, the two substrates of the enzyme bind at opposite sides of the catalytic  residue.
 * '''carnitine binding site'''
-The carnitine substrate has to be positioned in a way that the proton of its hydroxyl group can interact with the nitrogen N3 of the catalytic residue histidine 343. Carnitine binding in the right position is made possible by electrostatic interactions and the formation of hydrogen bondings between the '''carboxylate group''' of carnitine and residues near the active site. The residues that form the carnitine binding site can be found in the C domain and in the N domain.
+The carnitine substrate has to be positioned in a way that the proton of its hydroxyl group can interact with the nitrogen N3 of the catalytic residue histidine 343. Carnitine binding in the right position is made possible by electrostatic interactions and the formation of hydrogen bondings between the '''carboxylate group''' of carnitine and active site residues. Residues that form the carnitine binding site can be found in the C domain and in the N domain.
 The main <scene name='Sandbox_212/Hydrogen_bonding_residues/3'>residues involved in  hydrogen-bonding interactions </scene> are  <font color='#0000FF'>'''tyrosine452'''</font>, <font color='#90E050'>'''serine454'''</font>, and <font color='#FF0D0D'>'''threonine465'''</font>. They possess side chain hydroxyls which can interact with the carboxylic oxygen atoms of carnitine. One of the carboxylic oxygen atoms is also hydrogen-bonded to a <font color='#00007C'>'''water molecule '''</font><ref name="structure">PMID: 12526798</ref> Electrostatic interactions are formed by the carboxylate group of carnitine with the side chain guanidinium group of an arginine residue.
 The exact role of the '''trimethylammonium group''' during carnitine binding hasn’t been fully revealed yet. Carnitine is rather required for catalysis than for binding. Even though the trimethylammonium group has a positive charge on its nitrogen it is not surrounded by negatively charged residues which could balance it. Instead, the trimethylammonium group is situated in a rather hydrophobic environment.
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 ==Catalytic Mechanism of Carnitine Acyltransferases==
-[[Image:Machanism fatty acid transfer.jpg|thumb|700px|left]]
+[[Image:Mechanism fatty acid transfer.jpg|thumb|catalytic mechanism of fatty acid transfer|700px|left]]
 It is assumed that the whole family of carnitine acyltransferases share the same catalytic mechanism, because certain residues in the catalytic side (histidine343, serine554) are conserved throughout the family.
-Histidine 343 is probably the most important residue in catalysis.  First, it induces optimal substrate binding by forming a hydrogen bond between the hydrogen of the substrate’s reactive group and its side chain. After all substrate attained the right position, the catalytic histidine residue is ready to extract a proton from either the hydroxyl group of carnitine or the thiol group of CoA. Thus the catalytic histidine residue can be considered as a general base in catalysis.Which proton is extracted depends on the direction of the reaction. Acyl- carnitine is formed by extracting a proton from carnitine, whereas acyl-CoA is formed by extracting a proton from CoA.
+Histidine 343 is probably the most important residue in catalysis.  First, it induces optimal substrate binding by forming a hydrogen bond between its side chain and the hydrogen atom of the substrate’s reactive group. As soon as all substrates attained the right position, the catalytic histidine residue is ready to extract a proton from either the hydroxyl group of carnitine or the thiol group of CoA. The catalytic histidine residue can be considered as a general base in catalysis.Which proton is extracted depends on the direction of the reaction. Acyl- carnitine is formed by extracting a proton from carnitine, whereas acyl-CoA is formed by extracting a proton from CoA.
-The extraction of a hydrogen atom leads to the development of a tetrahedral oxyanion intermediate. This oxyanion is stabilized by the side chain hydroxyl of serine 554 through hydrogen bonding as well as  by the positive charge on the trimethylammonium group of carnitine. Since the positive charge of the carnitine substrate is necessary for the carnitine acyltransferase mediated reaction to happen, this catalysis can be described as '''substrate-assisted catalysis'''.<ref>PMID:15591000</ref>
+The extraction of a hydrogen atom leads to the development of a tetrahedral <font color='#175487'>'''oxyanion'''</font> <font color='#E8B613'>'''intermediate'''</font>. This oxyanion is stabilized by the <font color='#A62929'>'''side chain hydroxyl of serine 554'''</font> through hydrogen bonding as well as  by the <font color='#A62929'>'''positive charge on the trimethylammonium group'''</font>  of carnitine. As the positive charge of the carnitine substrate is necessary for the carnitine acyltransferase mediated reaction to happen, this catalysis can be described as '''substrate-assisted catalysis'''.<ref name="structure1">PMID:15591000</ref>
 == Regulation ==
 One of the most common regulation systems of carnitine acyltransferases involves inhibition by malonyl-CoA, an intermediate in the synthesis of fatty acids. Malonyl-CoA inhibits long-chain carnitine acyltransferase activity by all three enzymes at similar concentrations in the physiological range. Moreover, the mitochondrial ( CAT) and peroxisomal (COT) enzymes can also be regulated through mRNA transcription by a number of shared factors. Although the microsomal enzyme is less well studied, there does, indeed, appear to be a pattern of coordinate regulation for this system.