chain oxygen atom of Asp-375 deprotonating acetyl CoA's alpha carbon atom to form an enolate anion which in turn is neutralized by protonation by His-274 to form an enol intermediate [H2C=C(OH)−SCoA]. At this point, the epsilon nitrogen lone pair of electrons on His-274 formed in the last step abstracts the hydroxyl enol proton to reform an enolate anion that initiates a nucleophilic attack on the oxaloacetate's carbonyl carbon [−O2CCH2C(=O)CO2−] which in turn deprotonate the epsilon nitrogen atom of His-320. This nucleophilic addition results in the formation of citroyl−CoA [−O2CCH2CH(CO2−)CH2C(=O)−SCoA]. At this point, a water molecule is deprotonated by the epsilon nitrogen atom of His-320 and hydrolysis is initiated. One of the oxygen's lone pairs nucleophilically attacks the carbonyl carbon of citroyl−CoA. This forms a tetrahedral intermediate and results in the ejection of −SCoA as the carbonyl reforms. The −SCoA is protonated to form HSCoA. Finally, the hydroxyl added to the carbonyl in the previous step is deprotonated and citrate [−O2CCH2C(OH)(CO2−)CH2CO2−] is formed. Inhibition The enzyme is inhibited by high ratios of ATP:ADP and NADH:NAD, as high concentrations of ATP and NADH show that the energy supply is high for the cell. It is also inhibited by succinyl-CoA and propionyl-CoA, which resembles Acetyl-coA and acts as a competitive inhibitor to acetyl-CoA and a noncompetitive inhibitor to oxaloacetate. Citrate inhibits the reaction and is an example of product inhibition. The inhibition of citrate synthase by acetyl-CoA analogues has also been well documented and has been used to prove the existence of a single active site. These experiments have revealed that this single site alternates between two forms, which participate in ligase and hydrolase activity respectively. This protein may use the morpheein model of allosteric regulation. References External links PDBe-KB provides an overview of all the structure information available in the PDB for Human Citrate synthase, mitochondrial Cell biology EC 2.3.3 Mitochondrial proteins