Citrate Synthase

From Proteopedia

The Structure and Mechanism of Citrate Synthase

Template:STRUCTURE 1ctsCitrate 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.

Structure: Citrate synthase exists as a homodimer. Each identical subunit is comprised almost entirely of α helices (making it an all α protein) and consists of a large and a small domain. In its free enzyme state, citrate synthase exists in “open” form, with its two domains forming a cleft containing the substrate binding site. When substrate (oxaloacetate) binds, the smaller domain undergoes an 18° rotation, sealing the oxaloacetate binding site and resulting in the “closed” conformation. This conformational change not only prevents solvent from reaching the bound oxaloacetate, 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, ionizable side chains of citrate synthase participate in acid-base catalysis: His 274, His 320, and Asp 375. First, Asp 375 (a base) removes a proton from the methyl group of acetyl-CoA to form its enol. His 274 stabilizes the acetyl-CoA enolate by forming a hydrogen bond with the enolate oxygen. The enolate then nucleophilically attacks oxaloacetate’s carbonyl carbon, and His 320 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.