Citrate Synthase

The Structure and Mechanism of Citrate SynthaseThe Structure and Mechanism of Citrate Synthase

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

spinqualitylabelsall models Citrate Synthase Open Form 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 (in fact, it is often used as a quantitative enzyme marker for intact mitochondria), it is encoded by nuclear DNA, not mitochondrial . 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: Citrate synthase is a single amino acid chain . Biologically, however, it exists as a . 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) . When oxaloacetate binds, the smaller domain undergoes an 18° rotation, sealing the oxaloacetate binding site and resulting in the (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 .

Mechanism: The reaction mechanism for citrate synthase was proposed by James Remington. In this mechanism, three ionizable side chains in the of citrate synthase participate in acid-base catalysis: His 274, His 320, and Asp 375. First, (a base) removes 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 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.

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) .