Citrate Synthase

Citrate synthase 'closed' form (6cts) and the reaction

The Structure and Mechanism of Citrate Synthase

spinqualitylabelsall models Citrate Synthase Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D

Citrate synthase is an enzyme active in all examined cells, where it is most often responsible for catalyzing the first reaction of the 3cts: 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. The standard free energy change (ΔG°’) for the citrate synthase reaction is -31.5kJ/mol . 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.

Structure: Biologically, citrate synthase exists as a homodimer of a single amino acid chain monomer. 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 an “open” form of the homodimer, with its two domains forming a cleft containing the substrate (oxaloacetate) binding site (PDB: 1cts) . When oxaloacetate binds, the smaller domain undergoes an 18° rotation, sealing the oxaloacetate binding site and resulting in the closed conformation of the homodimer (PDB: 2cts). The dramatic conformational change is best illustrated via a morph between the "open" and "closed" states, and be sure to view the morph from the side 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 .

spinqualitylabelsall models Citrate synthase catalysis in the closed conformation Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Mechanism: Three side chains in each of the two active sites 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 these three side chains and the two substrates are together in an arrangement favorable for reaction. (By contrast, the active site residues are significantly farther apart 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 and is illustrated here in three dimensions using structures resembling key states of the reaction. In this mechanism, three ionizable side chains in the active site of citrate synthase participate in acid-base catalysis: His 274, His 320, and Asp 375. 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, Asp 375 acts as a base removing a proton from the methyl group of acetyl-CoA, 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 enol of acetyl-CoA then nucleophilically attacks 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 citryl-CoA 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 a nearby water is deprotonated by His 320 and the oxygen attacks citryl-CoA. Release of the CoA and the citrate involves return of the closed conformation 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, citrate (which competes with oxaloacetate), and succinyl-CoA (an example of competitive feedback inhibition) . In many plants, bacteria and fungi, such as the peroxisomes of baker's yeast, citrate synthase plays a role in the glyoxylate cycle.

The reaction mechanism for catalysis by citrate synthase

spinqualitylabelsall models Citrate Synthase Closed Form (Monomer), 2cts Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D

spinqualitylabelsall models Citrate Synthase Open Form (Monomer), 1cts Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D

3D structures of Citrate Synthase

Update June 2011

3msu – CitS – Francisella tularensis
3enj – CitS – Wild boar
2p2w – CitS – Thermotoga maritima
2c6x – CitS residues 2-364 – Bacillus subtilis
3l96, 1owc, 1nxe – EcCitS (mutant) – Eschericia coli
1k3p – EcCitS II
2ibp – CitS – Pyrobaculum aerophilum
1iom, 1ixe – CitS – Thermus thermophilus
1o7x – CitS – Sulfolobus solfataricus
1a59 – CitS – Antarctic bacterium
1aj8 – CitS - Pyrococcus furiosus
5csc, 3cts – cCitS – chicken
1cts, 2cts – pCitS - pig

Citrate synthase binary complex

3l97 - EcCitS (mutant) + S-carboxymethyl-CoA
2r9e – TaCitS + citryl dethia CoA – Thermoplasma acidophilum
6cts – cCitS + citryl thioether CoA
3l98, 1owb, 1nxg - EcCitS (mutant) + NADH
3l99 - EcCitS (mutant) + oxaloacetate
2ifc - TaCitS + oxaloacetate

Citrate synthase ternary complex

2r26 - TaCitS + oxaloacetate + S-carboxymethyl-CoA
2h12 - CitS + oxaloacetate + carboxymethyl dethia-CoA – Acetobacter aceti
1al6, 1csr, 1css, 1csh, 1csi - cCitS + oxaloacetate + carboxymethyl dethia-CoA derivative
5cts - cCitS + oxaloacetate + carboxymethyl CoA
1amz - cCitS + malate + nitromethyl dethia-CoA
1csc, 2csc, 3csc, 4csc - cCitS + malate + carboxymethyl CoA
6csc - cCitS + citrate + trifluoroacetonyl-CoA
4cts - pCitS + oxaloacetate + S-acetonyl CoA

See Also

• The Citric Acid Cycle
• Carbohydrate Metabolism

External Resources

• Citrate Synthase: September 2007 Molecule of the Month as part of the series of tutorials that are at the RCSB Protein Data Bank and written by David Goodsell
• An interactive schematic animation of Citrate synthase's reaction mechanism from Lehninger's Principles of Biochemistry
• Citrate Synthase at Wikipedia

Proteopedia Page Contributors and Editors (what is this?)

Wayne Decatur, Daniel Eddelman, David Canner, Angel Herraez, Eric Martz, Joel L. Sussman, Alexander Berchansky, Michal Harel