Citrate synthase is a very important catalyst, critical to the initiation of the citric acid cycle.
Citrate synthase (1) has been found in nearly all living cells. It is primarily found within the inner membrane-matrix fraction of mitochondria in eukaryotic cells. Synthesis of citrate synthase occurs in the cytosol and is then transported into the mitochondria (2).
Citrate synthase is key within the citric acid metabolic pathway. Citrate synthase reaction mechanism (3).
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The citric acid cycle starts with a condensation reaction of the four-carbon oxaloacetate with the two-carbon acetyl group of acetyl CoA. Oxaloacetate reacts with acetyl CoA and water to give citrate and CoA as products. This reaction, an aldol condensation followed by a hydrolysis, is catalyzed by citrate synthase. Oxaloacetate first forms citryl CoA after a condensation with acetyl CoA, which is followed by hydrolysis yielding citrate and CoA. The hydrolysis of citryl CoA, thioester intermediate high in energy, drives the overall reaction to favor the product citrate. In essence, the hydrolysis of the thioester powers the synthesis of a new molecule from two precursors. It is important to minimize any side reactions, because the above reaction initiates the citric acid cycle. Citrate synthase prevents wasteful processes such as the hydrolysis of acetyl CoA (4).
Citrate synthase exhibits sequential, ordered kinetics: oxaloacetate binds first, followed by acetyl CoA. This is due to a major conformational rearrangement induced by oxaloacetate resulting in the formation of a binding site for acetyl CoA. The binding of oxaloacetate transforms the open form of citrate synthase observed in the absence of ligands to a closed form. Wasteful hydrolysis of acetyl CoA is avoided since citrate synthase does not hydrolyze acetyl CoA well. Acetyl CoA also does not bind to citrate synthase until oxaloacetate is bound and ready for condensation, and the catalytic residues crucial for hydrolysis of the thioester linkage are not appropriately positioned until citryl CoA is formed. An induced fit prevents an undesirable side reaction. Once Coenzyme A leaves the citrate synthase, citrate also leaves, and citrate synthase reverts back to the open conformation (4).
The structure of citrate synthase is predominantly alpha helices (20 in each subunit) arranged more or less in layers, each layer consisting of approximately antiparallel pairs of helices. A small section of anti-parallel beta sheets on each subunit also exist, all connected with numerous turns, denoted in white and blue < > (2). Citrate synthase is in the open conformation before the ligand binding.
Citrate synthase is a homodimer with two 49-kd subunits, composed of two identical polypeptide chain each 437 amino acids in length< >. Each dimer of citrate synthase has a molecular weight of 100,000 (5).
Subunit A of the dimer comprises of a large and small domain, which is identical to the large and small domains of the B subunit < >. The large domains form the sites of interaction of the two subunits in the dimeric molecule.
Please click < > to load open conformation of citrate synthase. A space-filled model exemplifies the open nature of the unbound molecule < >. When oxaloacetate binds to citrate synthase, the molecule makes a dramatic conformational change. Please click < > to load closed conformation of citrate synthase. Compare the dramatic difference between the space-filled model of the open conformation of citrate synthase with the closed conformation < >.
Oxaloacetate binds to the citrate synthase < > at the critical active site residues < > His 274, His 320, and Asp 375. Other residues also involved in the binding of oxaloacetate < > are His 238 , Arg 329, Arg 401 (5).
Recently, a method was developed to identify residues that are involved in inducing closure in enzymes when an open unliganded structure and a close liganded structure are available. Using this method, three potential "closure-inducing residues" have been identified in citrate synthase: His 274, His 320, and Arg 329. These residues interact with oxaloacetate to induce closure in citrate synthase (6). Arg 329, which is situated at the base of the alpha helix residues 328-341< >, forms a strong salt-bridge with oxalacetate, and this interaction is believed to help citrate synthase overcome the energy barrier in moving from the open to the fully closed state (6).
Mechanism of synthesis of citryl CoA by citrate synthase(4).
Click to reload citrate synthase. Click < > to go highlight the active site of citrate synthase with oxaloacetate. Coenzyme A (CoA) and citrate now bind to citrate synthase after oxaloacetate is bound < >.
Citrate synthase catalyzes the condensation reaction (refer to the mechanism chart above) by orienting substrates close together and polarizing certain bonds. His 274 gives off a proton to the acetyl CoA carbonyl oxygen, which results in the removal of a methyl proton by Asp 375. Oxaloacetate is then activated by a proton transfer from His 320 to its carbonyl carbon. The attack by the enol of acetyl CoA on the oxaloacetate carbonyl carbon results in the formation of a carbon-carbon bond. The resulting citryl CoA that is produced further induces structural changes in the enzyme, completely enclosing the active site. His 274 donates another proton to hydrolyze the thioester. CoA leaves the citrate synthase, followed by citrate. The enzyme then returns to the "S-shaped" initial open conformation unbound by ligands.
1. Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (http://www.rcsb.org/pdb/). PDB: E.C. 4.1.3.7.
2. Remington, S., Wiegand, G., and Huber, R. 1982. Crystallographic Refinement and Atomic Models of Two Different Forms of Citrate Synthase at 2.7 and 1.7 Ǻ Resolution. J. Mol. Biol. 158: 111-152.
3. Miles, B. 2003. The Citric Acid Cycle. (http://www.tamu.edu/classes/bich/bmiles/lectures/CITRIC.pdf#search='miles%20citric%20acid%20cycle')
4. Berg, J.M., Tymoczko, J.L., and Stryer, L. 2002. Biochemistry, 5th ed. 472-473.
5. Wiegand, G., Remington, S., Deisenhofer, J., and Huber, R. 1984. Crystal Structure Analysis and Molecular Model of a Complex of Citrate Synthase with Oxaloacetate and S-Acetonyl-coenzyme A. J. Mol. Biol. 174: 205-219.
6. Daidone, I., Roccatano, D., and Hayward, S. 2004. Investigating the Accessibility of the Closed Domain Conformation of Citrate Synthase using Essential Dynamics Sampling. J. Mol. Biol. 339: 515-525.