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==The Structure and Mechanism of Citrate Synthase==
{{BAMBED
<applet load='2cts' size='300' color='white' frame='true' align='right' caption='Citrate Synthase Closed Form' name='closed'/> <applet load='1cts' size='300' color='white' frame='true' align='right' caption='Citrate Synthase Open Form' name='open'/>
|DATE=March 3, 2011
Citrate synthase is an enzyme active in the mitochondria, where it is responsible for catalyzing the first reaction of the citric acid cycle (Krebs Cycle): the condensation of acetyl-CoA and oxaloacetate to form citrate.  The standard free energy change (ΔG°’) for the citrate synthase reaction is -31.5kJ/mol <ref name="voet">Voet, Donald, Judith G. Voet, and Charlotte W. Pratt. Fundamentals of Biochemistry: Life at the Molecular Level. Hoboken, NJ: Wiley, 2008.</ref>. This negative free energy value means that citrate synthase is likely to function far from equilibrium under physiological conditions, and is thus a rate-determining enzyme in the citric acid cycle.
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|BAMBEDDOI=10.1002/bmb.20519
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<StructureSection load='1cts' size='350' side='right' scene='User:Wayne_Decatur/1cts_to_2cts_(citrate_synthase)_morph_methods/Camorph/5' caption='Open conformation of citrate synthase dimer complex with citrate (PDB code [[1cts]]) and closed conformation of citrate synthase dimer complex with citrate and CoA (PDB code [[2cts]])'>
 
==Overview==
 
'''Citrate synthase''' is an enzyme active in all examined cells, where it is most often responsible for catalyzing the first reaction of the [[The Citric Acid Cycle|citric acid cycle (Krebs Cycle or the tricarboxylic acid <nowiki>[</nowiki>TCA<nowiki>]</nowiki> cycle)]]: the condensation of acetyl-CoA and oxaloacetate to form citrate.  Although in eukaryotes it is a mitochondrial enzyme, and in fact, is often used as a enzyme marker for intact mitochondria, it is encoded by nuclear DNA<ref>[http://en.wikipedia.org/wiki/Citrate_synthase "Citrate Synthase -." Wikipedia, the Free Encyclopedia. Web. 22 Mar. 2010].</ref>. The standard free energy change (ΔG°’) for the citrate synthase reaction is
-31.5kJ/mol <ref name="voet">Voet, Donald, Judith G. Voet, and Charlotte W. Pratt. Fundamentals of Biochemistry: Life at the Molecular Level. Hoboken, NJ: Wiley, 2008.</ref>. This negative free energy value means that citrate synthase is likely to function far from equilibrium under physiological conditions, and is thus a rate-determining enzyme in the citric acid cycle. See also:<br />
*[[Krebs cycle carbons]]
*[[Krebs cycle importance]]
*[[Krebs cycle overview]]
*[[Krebs cycle reactions]]
*[[Citric Acid Cycle]]
*[[Krebs cycle step 1]]
*[[Glyoxylate cycle]]
 
'''2-methylcitrate synthase''' catabolizes propionate to succinate and pyruvate<ref>PMID:9579066</ref>.
 
==Structure==
    
    
'''Structure:''' Citrate synthase is a single amino acid chain <scene name='Daniel_Eddelman_Sandbox_2/Cts_open_monomer/1'>monomer</scene>.  Biologically, however, it exists as a  
Biologically, citrate synthase exists as a <scene name='Daniel_Eddelman_Sandbox_2/Cts_open_monomer/2'>homodimer</scene> of a single amino acid chain <scene name='Daniel_Eddelman_Sandbox_2/Cts_open_monomer/1'>monomer</scene>. Each identical subunit consists of a large and a small domain, and is comprised almost entirely of α helices (making it an all α protein).  In its free enzyme state, citrate synthase exists in <scene name='Daniel_Eddelman_Sandbox_2/Cts_open_monomer/2'>an “open” form of the homodimer</scene>, with its two domains forming a cleft containing the substrate (oxaloacetate) binding site (PDB: [[1cts]]) <ref name="1cts">PMID:7120407</ref><ref name="substrate note">In this structure [[1cts]], citrate, the resulting product of the conversion, is actually bound where oxaloacetate binds.</ref>.  When oxaloacetate binds, the smaller domain undergoes an 18° rotation, sealing the oxaloacetate binding site<ref>PMID:7308213</ref> and resulting in the <scene name='Daniel_Eddelman_Sandbox_2/Closed_homodimer/1'>closed conformation of the homodimer</scene> (PDB: [[2cts]])<ref name="1cts" />.  The dramatic conformational change is best illustrated via <scene name='User:Wayne_Decatur/1cts_to_2cts_(citrate_synthase)_morph_methods/Camorph/5'>a morph between the "open" and "closed" states</scene>, and be sure to view <scene name='User:Wayne_Decatur/1cts_to_2cts_(citrate_synthase)_morph_methods/Camorphside/3'>the morph from the side</scene> as well to get a full sense of the structural change. The conformational change not only prevents solvent from reaching the bound substrate, but also generates the acetyl-CoA binding site.  This presence of “open” and “closed” forms results in citrate synthase having Ordered Sequential kinetic behavior <ref name="voet" />.<br>
<scene name='Daniel_Eddelman_Sandbox_2/Cts_open_monomer/2'>homodimer</scene>. Each identical subunit consists of a large and a small domain, and is comprised almost entirely of α helices (making it an all α protein).  In its free enzyme state, citrate synthase exists in “open” form, with its two domains forming a cleft containing the substrate (oxaloacetate) binding site (PDB: [[1cts]]) <ref>PMID:7120407</ref>.  When oxaloacetate binds, the smaller domain undergoes an 18° rotation, sealing the oxaloacetate binding site and resulting in the <scene name='Daniel_Eddelman_Sandbox_2/Closed_homodimer/1' target='closed' >closed conformation</scene> (PDB: [[2cts]]).  This conformational change not only prevents solvent from reaching the bound substrate, but also generates the acetyl-CoA binding site.  This presence of “open” and “closed” forms results in citrate synthase having Ordered Sequential kinetic behavior <ref name="voet" />.
{{Button Toggle AnimationOnPause}}<br>
{{Link Toggle FancyCartoonHighQualityView}}


'''Mechanism:''' The reaction mechanism for citrate synthase was proposed by James Remington.  In this mechanism, three ionizable side chains in the
==Mechanism==
<scene name='Daniel_Eddelman_Sandbox_2/Cts_active_site/4' target='open' >active site</scene> of citrate synthase participate in acid-base catalysis: His 274, His 320, and Asp 375.  First, <scene name='Daniel_Eddelman_Sandbox_2/Asp_375/1' target='open' >Asp 375</scene> (a base) removes a proton from the methyl group of acetyl-CoA to form its enol.  <scene name='Daniel_Eddelman_Sandbox_2/His_274/1' target='open' >His 274</scene> stabilizes the acetyl-CoA enolate by forming a hydrogen bond with the enolate oxygen.  The enolate then nucleophilically attacks oxaloacetate’s carbonyl carbon, and
<scene name='Daniel_Eddelman_Sandbox_2/His_320/1' target='open' >His 320</scene> donates a proton to oxaloacetate’s carbonyl group in a concerted step, forming citryl-CoA (which remains bound to the enzyme).  Finally, citryl-CoA is hydrolyzed to citrate and CoA.


<scene name='Daniel_Eddelman_Sandbox_2/Open_default/1' target='open' >Open Default</scene>
<scene name='Citrate_Synthase/5ctsactivesitedimer/2'>Three side chains in each of the two active sites</scene> of the dimer contribute directly to the chemistry of catalysis. Focusing on a single active site in the closed conformation, one can easily observe that <scene name='Citrate_Synthase/5ctsactivesiteresidues/10'>these three side chains and the two substrates are together</scene> in an arrangement favorable for reaction. (By contrast, <scene name='Citrate_Synthase/Activesite1ctsto2cts/12'>the active site residues are significantly farther apart</scene> in the open conformation; the difference in the distance is ~5&Aring; along the axis that changes the most during the conformation shift.) {{Link Toggle AnimationOnPause}}
The reaction mechanism for citrate synthase was proposed by Remington and colleagues<ref name="1cts">PMID:7120407</ref><ref>PMID: 2337600</ref> and is illustrated here in three dimensions using structures resembling key states of the reaction<ref>[[5cts]] as the state preceding condensation with oxaloacetate and a non-reactive version of acetyl-CoA bound, [[6cts]] as the state containing the bound intermediate, and [[3cts]] as the complex with the products. Positions of hydrogens on the ligands were calculated and added back to structures in the reaction scheme for instructional purposes and are not present in the experimentally-determined structures; additionally, arrows are drawn with atoms of the analog of acetyl-CoA to approximate the position of the reactive groups only as the reactive groups are not actually part of the analog or the molecules would have reacted; please, see the reaction scheme on this page for a more thorough accounting of the chemistry.</ref>.  In this mechanism, three ionizable side chains in the active site of citrate synthase participate in acid-base catalysis: <scene name='Citrate_Synthase/5ctsactivesiteresidues/10'>His 274, His 320, and Asp 375</scene>. Citrate synthase is among one of the few enzymes that can directly form a carbon-carbon bond without the presence of metal ion cofactors.<br>
*In step one, <scene name='Citrate_Synthase/5ctsasp375/9'>Asp 375 acts as a base removing a proton from the methyl group of acetyl-CoA</scene>, resulting in acetyl-CoA forming its enol; His 274 (magenta) stabilizes the acetyl-CoA enol by forming a hydrogen bond with the enol's oxygen. (See the reaction scheme below for a more thorough accounting of the chemistry.)
*In step two (condensation), the <scene name='Citrate_Synthase/5ctscondensation/3'>enol of acetyl-CoA then nucleophilically attacks</scene> oxaloacetate’s carbonyl carbon, and His 320 (cyan) acts as an acid donating a proton to oxaloacetate’s carbonyl group in a concerted step, forming <scene name='Citrate_Synthase/6ctsactivesiteresidues/4'>citryl-CoA</scene> as acetyl-CoA and oxaloacetate become covalently linked; citryl-CoA remains bound to the enzyme at this step in the actual reaction although that linkage is not represented in the 3D structure.
*Finally, citryl-CoA is hydrolyzed to citrate and CoA as <scene name='Citrate_Synthase/6ctsactivehydrolysis/5'>a nearby water is deprotonated by His 320 and the oxygen attacks citryl-CoA</scene>. Release of the <scene name='Citrate_Synthase/3ctsproducts/2'>CoA and the citrate</scene> involves return of the <scene name='Citrate_Synthase/3ctsproductssf/2'>closed conformation</scene> to the open conformation.<br/>


<scene name='Daniel_Eddelman_Sandbox_2/Closed_default/1' target='closed' >Closed Default</scene>
{{Button HeteroCPKPLUSCSColorScheme}} {{Link Toggle FancyCartoonHighQualityView}}
 
==Regulation==
 
Perhaps the most crucial regulators of the citrate synthase reaction are its substrates, acetyl-CoA and oxaloacetate.  Both are present in the mitochondria at concentrations below saturation of citrate synthase.  The metabolic flux is controlled by substrate availability, so controlling the levels of acetyl-CoA and oxaloacetate in the mitochondria controls the rate of reaction.  Furthermore, citrate synthase is inhibited by NADH, <scene name='Citrate_Synthase/3ctscitrateonly/1'>citrate</scene> (which competes with oxaloacetate), and succinyl-CoA (an example of competitive feedback inhibition) <ref>PMID:3013232</ref>. In many plants, bacteria and fungi, such as the peroxisomes of baker's yeast, citrate synthase plays a role in the glyoxylate cycle<ref>PMID:3023912</ref><ref>PMID: 2181273</ref><ref>PMID:17615299</ref>.
*<scene name='Citrate_Synthase/Cv/1'>Citrate Synthase Closed Form (Monomer)</scene> [[2cts]]
*<scene name='Citrate_Synthase/Cv/2'>'Citrate Synthase Open Form (Monomer)</scene> [[1cts]]
 
==3D structures of Citrate Synthase==
[[Citrate Synthase 3D structures]]
 
</StructureSection>
__NOTOC__
[[Image:CitSynReaction schematic as image.png|1024px|left|thumb| <span style="font-size:1.2em;">The reaction mechanism for catalysis by citrate synthase</span>]]
{{Clear}}
 
[[Image:2cts plus overall reaction.png|500px|left|thumb| <span style="font-size:1.2em;">Citrate synthase 'closed' form complex with CoA and citrate ([[2cts]]) and the reaction</span>]]
{{Clear}}
 
==See Also==
* [[The Citric Acid Cycle]]
* [[Carbohydrate Metabolism]]
* [[Krebs cycle step 1]]
==Literature and Notes==
<references/>
<references/>
==External Resources==
*[http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb93_1.html Citrate Synthase: September 2007 Molecule of the Month] as part of the series of tutorials that are at [http://www.pdb.org/pdb/home/home.do the RCSB Protein Data Bank] and written by [[User:David_S._Goodsell|David Goodsell]]
*[http://bcs.whfreeman.com/lehninger/pages/bcs-main.asp?s=00010&n=16000&i=16010.01&v=category&o=|00510|00520|00530|00540|00550|00PRS|00010|00020|00030|00040|00050|00060|00070|00080|00090|00100|00110|00120|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|24000|25000|26000|27000|28000|99000|&ns=0&t=&uid=0&rau=0 An interactive schematic animation of Citrate synthase's reaction mechanism from Lehninger's Principles of Biochemistry]
*[http://en.wikipedia.org/wiki/Citrate_synthase Citrate Synthase at Wikipedia]
[[Category:Topic Page]]
[[Category:Featured in BAMBED]]

Latest revision as of 11:59, 11 January 2023

This page, as it appeared on March 3, 2011, was featured in this article in the journal Biochemistry and Molecular Biology Education.


Overview

Citrate synthase is an enzyme active in all examined cells, where it is most often responsible for catalyzing the first reaction of the citric acid cycle (Krebs Cycle or the tricarboxylic acid [TCA] cycle): the condensation of acetyl-CoA and oxaloacetate to form citrate. Although in eukaryotes it is a mitochondrial enzyme, and in fact, is often used as a enzyme marker for intact mitochondria, it is encoded by nuclear DNA[1]. The standard free energy change (ΔG°’) for the citrate synthase reaction is

-31.5kJ/mol [2]. This negative free energy value means that citrate synthase is likely to function far from equilibrium under physiological conditions, and is thus a rate-determining enzyme in the citric acid cycle. See also:

2-methylcitrate synthase catabolizes propionate to succinate and pyruvate[3].

Structure

Biologically, citrate synthase exists as a of a single amino acid chain . Each identical subunit consists of a large and a small domain, and is comprised almost entirely of α helices (making it an all α protein). In its free enzyme state, citrate synthase exists in , with its two domains forming a cleft containing the substrate (oxaloacetate) binding site (PDB: 1cts) [4][5]. When oxaloacetate binds, the smaller domain undergoes an 18° rotation, sealing the oxaloacetate binding site[6] and resulting in the (PDB: 2cts)[4]. The dramatic conformational change is best illustrated via , and be sure to view as well to get a full sense of the structural change. The conformational change not only prevents solvent from reaching the bound substrate, but also generates the acetyl-CoA binding site. This presence of “open” and “closed” forms results in citrate synthase having Ordered Sequential kinetic behavior [2].


Mechanism

of the dimer contribute directly to the chemistry of catalysis. Focusing on a single active site in the closed conformation, one can easily observe that in an arrangement favorable for reaction. (By contrast, in the open conformation; the difference in the distance is ~5Å along the axis that changes the most during the conformation shift.)

The reaction mechanism for citrate synthase was proposed by Remington and colleagues[4][7] and is illustrated here in three dimensions using structures resembling key states of the reaction[8]. In this mechanism, three ionizable side chains in the active site of citrate synthase participate in acid-base catalysis: . Citrate synthase is among one of the few enzymes that can directly form a carbon-carbon bond without the presence of metal ion cofactors.

  • In step one, , resulting in acetyl-CoA forming its enol; His 274 (magenta) stabilizes the acetyl-CoA enol by forming a hydrogen bond with the enol's oxygen. (See the reaction scheme below for a more thorough accounting of the chemistry.)
  • In step two (condensation), the oxaloacetate’s carbonyl carbon, and His 320 (cyan) acts as an acid donating a proton to oxaloacetate’s carbonyl group in a concerted step, forming as acetyl-CoA and oxaloacetate become covalently linked; citryl-CoA remains bound to the enzyme at this step in the actual reaction although that linkage is not represented in the 3D structure.
  • Finally, citryl-CoA is hydrolyzed to citrate and CoA as . Release of the involves return of the to the open conformation.

Regulation

Perhaps the most crucial regulators of the citrate synthase reaction are its substrates, acetyl-CoA and oxaloacetate. Both are present in the mitochondria at concentrations below saturation of citrate synthase. The metabolic flux is controlled by substrate availability, so controlling the levels of acetyl-CoA and oxaloacetate in the mitochondria controls the rate of reaction. Furthermore, citrate synthase is inhibited by NADH, (which competes with oxaloacetate), and succinyl-CoA (an example of competitive feedback inhibition) [9]. In many plants, bacteria and fungi, such as the peroxisomes of baker's yeast, citrate synthase plays a role in the glyoxylate cycle[10][11][12].

3D structures of Citrate Synthase

Citrate Synthase 3D structures


Open conformation of citrate synthase dimer complex with citrate (PDB code 1cts) and closed conformation of citrate synthase dimer complex with citrate and CoA (PDB code 2cts)

Drag the structure with the mouse to rotate
The reaction mechanism for catalysis by citrate synthase
Citrate synthase 'closed' form complex with CoA and citrate (2cts) and the reaction

See AlsoSee Also

Literature and NotesLiterature and Notes

  1. ↑ "Citrate Synthase -." Wikipedia, the Free Encyclopedia. Web. 22 Mar. 2010.
  2. ↑ 2.0 2.1 Voet, Donald, Judith G. Voet, and Charlotte W. Pratt. Fundamentals of Biochemistry: Life at the Molecular Level. Hoboken, NJ: Wiley, 2008.
  3. ↑ Gerike U, Hough DW, Russell NJ, Dyall-Smith ML, Danson MJ. Citrate synthase and 2-methylcitrate synthase: structural, functional and evolutionary relationships. Microbiology (Reading). 1998 Apr;144 ( Pt 4):929-935. doi: , 10.1099/00221287-144-4-929. PMID:9579066 doi:http://dx.doi.org/10.1099/00221287-144-4-929
  4. ↑ 4.0 4.1 4.2 Remington S, Wiegand G, Huber R. Crystallographic refinement and atomic models of two different forms of citrate synthase at 2.7 and 1.7 A resolution. J Mol Biol. 1982 Jun 15;158(1):111-52. PMID:7120407
  5. ↑ In this structure 1cts, citrate, the resulting product of the conversion, is actually bound where oxaloacetate binds.
  6. ↑ Bayer E, Bauer B, Eggerer H. Evidence from inhibitor studies for conformational changes of citrate synthase. Eur J Biochem. 1981 Nov;120(1):155-60. PMID:7308213
  7. ↑ Karpusas M, Branchaud B, Remington SJ. Proposed mechanism for the condensation reaction of citrate synthase: 1.9-A structure of the ternary complex with oxaloacetate and carboxymethyl coenzyme A. Biochemistry. 1990 Mar 6;29(9):2213-9. PMID:2337600
  8. ↑ 5cts as the state preceding condensation with oxaloacetate and a non-reactive version of acetyl-CoA bound, 6cts as the state containing the bound intermediate, and 3cts as the complex with the products. Positions of hydrogens on the ligands were calculated and added back to structures in the reaction scheme for instructional purposes and are not present in the experimentally-determined structures; additionally, arrows are drawn with atoms of the analog of acetyl-CoA to approximate the position of the reactive groups only as the reactive groups are not actually part of the analog or the molecules would have reacted; please, see the reaction scheme on this page for a more thorough accounting of the chemistry.
  9. ↑ Wiegand G, Remington SJ. Citrate synthase: structure, control, and mechanism. Annu Rev Biophys Biophys Chem. 1986;15:97-117. PMID:3013232 doi:http://dx.doi.org/10.1146/annurev.bb.15.060186.000525
  10. ↑ Kim KS, Rosenkrantz MS, Guarente L. Saccharomyces cerevisiae contains two functional citrate synthase genes. Mol Cell Biol. 1986 Jun;6(6):1936-42. PMID:3023912
  11. ↑ Lewin AS, Hines V, Small GM. Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal. Mol Cell Biol. 1990 Apr;10(4):1399-405. PMID:2181273
  12. ↑ Lee YJ, Hoe KL, Maeng PJ. Yeast cells lacking the CIT1-encoded mitochondrial citrate synthase are hypersusceptible to heat- or aging-induced apoptosis. Mol Biol Cell. 2007 Sep;18(9):3556-67. Epub 2007 Jul 5. PMID:17615299 doi:10.1091/mbc.E07-02-0118

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