On the action of carboxy groups in the citrate synthase reaction

The aza analogue (RS)-3-hydroxy-2,5-pyrrolidinedione-3-acetic acid (6) of the five-membered citric anhydride (2) was prepared in the sequence citric acid----2-phenyl-1,3-dioxolan-4-one-5,5-diacetic acid (1)----citric acid beta-amide (3)----6 and used to resolve ambiguities in the mechanism of the citrate synthase reaction. The results yield no indication for the formation of anhydride 2 on the enzyme and favour the direct hydrolysis of the intermediate (3S)-citryl-CoA. Ammonolysis of the dioxolanone 1 in the reaction sequence described above produced not only citric acid beta-amide but also the alpha-isomer. This is shown to originate in the transient formation of anhydride 2. Hydrolysis of the dioxolanone 1 under "physiological conditions" occurs via anhydride 2, generated in intramolecular bifunctional catalysis by a protonated and a deprotonated carboxyl group. The catalytic residue Asp375 of citrate synthase is considered to operate on the enzyme as does the protonated carboxyl group in the chemical reaction and to generate enolic acetyl-CoA in cooperative catalysis with His274. This reaction of Asp375 may also facilitate the hydrolysis of citryl-CoA.