Citrate synthase (1CTS), originating in Sus scrofa, is a member of the Oxo Acid Lyase family (1). Citrate synthase participates in the citric acid cycle (otherwise known as the TCA cycle), which follows glycolysis in aerobic organisms. In this cycle, acetate (as acetyl-coA) is successively oxidized to form carbon dioxide, driving the release of reducing agents NADH and FADH2 (3). Citrate synthase acts in the first step of the citric acid cycle by catalyzing the condensation reaction between acetate (in the form of acetyl-CoA) and oxaloacetate to produce citrate, a six-carbon intermediate (7). Given that the citric acid cycle takes place in the mitochondria, citrate synthase is found in the mitochondrial matrix of eukaryotes, although it is encoded by nuclear DNA. It is translated in the cytoplasm as a precursor and transported into the mitochondria where it is localized to the inner membrane (8). The citric acid cycle is a necessary component of aerobic cellular respiration, and as a result citrate synthase is found ubiquitously in all aerobic organisms. Citrate synthase acts as the inducer of the citric acid cycle and proceeds forward irreversibly in standard conditions due to the strong negative free energy change of citrate synthesis (ΔG°’ = -31.4 kJ), triggering the entire cycle (4).
The citrate synthase enzyme found in animals, plants, fungi and archaebacteria is a homodimer composed of two identical monomeric subunits of 437 amino acid residues each, and each subunit has a weight of 4.892 kDa and an isoelectric point (pI) of 7.01 (5). The combined weight of the two subunits in the homodimer is thus 9.784 kDa. Each subunit is comprised of a total of 20 alpha helices, which together make up 75% of the amino acid structure, while the remaining residues are largely unstructured, save one beta-sheet of 13 residues on each of the subunits (10). The entire enzyme packs together to form a dense globular molecule (5). The two subunits of the citrate synthase each have their own active site that function independently of one another, and the subunits are divided further into a small and large domain (7). Each large domain contains 15 alpha helices and each small domain contains the remaining five alpha helices. The two subunits of the dimer interface with one another via four pairs of helices (each pair composed of one helix from each subunit) that twist together to form a structure reminiscent of a beta sheet (10). Remington et. al conducted crystallographic refinement of citrate synthase and discovered two crystal forms that differ in the arrangement of the domains. The tetragonal form is “open,” with a large cleft between the small and large domains that allows access of substrate to the active site. The monoclinic form is “closed” due to an 18° rotation of the small domain inwards to close the cleft around the substrate. In the tetragonal form, oxaloacetate binds to the active site, triggering a conformational change to the monoclinic form that not only protects the substrate from solvent but also exposes the acetyl-coA binding site (8). Once the substrates are bound, three active-site side chains consisting of two histidine residues (His 274 and His 320) and one asparagine residue (Asp 375) perform acid-base catalysis of the condensation reaction, producing two molecules of citrate per protein dimer (7,8).
The mechanism of citrate synthesis proceeds first by the binding of oxaloacetate to the binding site residues His 274, His 320 and Asp 275, which when bound to oxaloacetate induce the enzyme’s conformation change from the open to the closed form (7). Another important residue in this transition from open to closed form is Arg 329, which forms a salt bridge with oxaloacetate and lowers the energy barrier for the conformation change (10). Once the conformational change is triggered, the small and large domains of the subunit close inwards via a hinge consisting of the main chain between the active site residue His 275 and the connecting Gly 275. Once the enzyme is in the closed form, acetyl-coA is allowed access to the binding site. The alpha carbon on acetyl-coA is activated for nucleophilic attack by the removal of a proton by Asp 375, causing acetyl-coA to form an enol. The enol is stabilized by the formation of a hydrogen bond between acetyl-coA’s oxygen molecule and His 274. The active acetyl-coA enol then nucleophilically attacks the carbonyl carbon on oxaloacetate, and in the same step His 320 acts as an acid and accepts a proton from oxaloacetate’s carbonyl oxygen, leading to the formation of the intermediate citryl-coA (4). Once citryl-coA is produced, hydrolysis of its thioester bond occurs via the deprotonation of a water molecule by His 320 and subsequent attack of the remaining hydroxide ion at the thioester carbon center, leading to the release of CoA, which accepts a proton from His 274. Once citrate is produced, it is released and the citrate synthase undergoes a conformation change to its open form. Each molecule of citrate synthase has two active sites and as a result, two molecules of citrate are produced at a time per protein dimer (4). In addition to this condensation reaction, citrate synthase is also capable of performing the reverse lyase reaction by binding citrate in the active site of its open form. Similar to the binding of oxaloacetate, the binding of citrate triggers a conformation change to the closed form, which provides a second biniding site for coenzyme A. Thus, lysis of citrate synthase in the presence of coenzyme A yields oxaloacetate and acetyl-coA.
Given the large negative free energy change of citrate synthase and its driving role in the citric acid cycle, current research of the enzyme is largely concerned with how it is regulated. This research has revealed several allosteric inhibitors that control the activity of citrate synthase, specifically NADH and ATP (both of which are products of the Citric acid cycle). These two molecules bind allosterically to citrate synthase and decrease its affinity for it substrates. A third molecule, succinyl-coA, regulates citrate synthase through competitive inhibition by binding to its active site, thus blocking access of the substrate to the binding site. Succinyl-coA is a product of a later step in the Citric acid cycle and it accumulation in the mitochondrial matrix indicates an abundance of electron carriers (in the form of NADH and FADH2) and hence free energy (in the form of ATP) in the cell, suggesting that catabolism can be halted. Thus, succinyl-coA serves as a competitive feedback inhibitor of citrate synthase by inhibiting over catalysis of the citric acid cycle and stopping further catabolism (8). Finally, citrase itself regulates the activity of citrate synthase by competing with oxaloacetate for the binding to the active site. Thus, the lyase reaction competes wtih the condensation reaction in citrae synthase (10).
The Oxo Acid Lyase family of proteins includes not only citrate synthase but also several other proteins with similar functions, including 2-methyl citrate synthase and ATP citrate synthase (6). Lyases include all proteins that catalyze the breaking of chemical bonds between two atoms by means other than hydrolysis. Oxo acid lyases are a subset of the carbon-carbon lyase subgroup (6) in that they catalyze the lysis of carbon-carbon bonds. Because they are specific to oxo acids, this means that these lyases break carbon-carbon bonds in acids containing an oxygen atom. In the case of citrate synthase, the reverse of its condensation reaction is a lyase reaction, as the six-carbon citrate can be cleaved to form acetyl-coA and oxaloacetate (8). 2-methyl citrate synthase’s function is similar to that of citrate synthase in that it is responsible for the synthesis of citrate. However, it is found only algae and gram-negative bacteria (10). Unlike citrate synthase, 2-methyl citrate synthase is hexameric. ATP citrate, on the other hand, functions similarly to the lyase reaction of citrate synthase in that it catalyzes the cleavage of citrate to yield acetyl-coA. However, it functions in the cytsol and is not a dimer but rather a tetramer (9).
Algorithmic analysis of citrate synthase based on both primary structure and tertiary structure returned many homologous proteins for comparison. The first tool used was the NCBI Protein-Specific Iterated Basic Local Assignment Search Tool (PSI BLAST), which compares the query amino acid sequence, in this case that of citrate synthase, against the sequences of known proteins and returns E scores based on their homology. Low E scores indicate greater sequence similarity between the two proteins, with E = 0 indicating a perfect match. Alternatively, the Dali server is a protein structure database that compares the query protein’s tertiary structure against those of other proteins to analyze for 3-D similarities. The Dali search returns structurally similar proteins and assigns them Z scores, with higher Z scores indicating more structural similarity. Using both PSI-BLAST and Dali to search for homologues, citrate synthase was found to be similar in both structure and amino acid sequence to the transferase protein 2-methyl citrate synthase. The E-score between the two proteins was found to be 3e-171 and the Z-score was 37.3 indicating high homology (1,3).