Citrate Synthase

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Contents 1 The Structure and Mechanism of Citrate Synthase 2 Literature and Notes 3 Details of Structures Featured 4 See Also 5 External Resources

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

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. Although 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.

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) ^[3][4]. When oxaloacetate binds, the smaller domain undergoes an 18° rotation, sealing the oxaloacetate binding site^[5] and resulting in the (PDB: 2cts)^[3]. 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].

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 Mechanism: of the dimer contribute directly to the chemistry of catalysis. Specifically in each active site when in the closed conformation, in an arrangement favorable for reaction. (By contrast, [more than ~5Å at the farthest point] in the open conformation.) The reaction mechanism for citrate synthase was proposed by Remington and colleagues[3][6] and is illustrated here in three dimensions using structures resembling key states of the reaction[7]. In this mechanism, three ionizable side chains in the active site of a monomer half of citrate synthase participate in acid-base catalysis: . First, acts as a base removing a proton from the methyl group of acetyl-CoA to form its enol. stabilizes the acetyl-CoA enolate by forming a hydrogen bond with the enolate oxygen. The enolate then nucleophilically attacks oxaloacetate’s carbonyl carbon, and acts as an acid donating 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. Releasing of the CoA and the citrate involves returning 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) [8].

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

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

Literature 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. ↑ ^{3.0 3.1 3.2} 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
4. ↑ In this structure 1cts, citrate, the resulting product of the conversion, is actually bound where oxaloacetate binds.
5. ↑ 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
6. ↑ 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
7. ↑ 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
8. ↑ 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

Details of Structures Featured

1cts is a 1 chain structure with sequence from Sus scrofa. Full crystallographic information is available from OCA.

2cts is a 1 chain structure of sequence from Sus scrofa. Full crystallographic information is available from OCA.

3cts is a 1 chain structure of sequence from Gallus gallus. Full crystallographic information is available from OCA.

5cts is a 1 chain structure of sequence from Gallus gallus. Full crystallographic information is available from OCA.

6cts is a 1 chain structure of sequence from Gallus gallus. Full crystallographic information is available from OCA.

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