Kinetics of the Inhibition of Citrate Synthase from Pig Heart by Substrate Analogues

1. The kinetics of inhibition of pig‐heart citrate synthase by the substrate analogues ATP, propionyl‐CoA, and 2‐oxoglutarate have been investigated. Evidence is presented that the citrate‐synthase‐catalyzed condensation reaction between acetyl‐CoA and oxaloacetate proceeds by an effectively ordered ternary‐complex mechanism with oxaloacetate adding first to the enzyme. 2. Inhibition of citrate synthase by 2‐oxoglutarate is competitive with oxaloacetate, and due to the formation of a binary complex with a stability constant of 0.19 mM −1 . Inhibition by ATP and propionyl‐CoA is competitive with acetyl‐CoA. ATP binds equally strongly (a binding constant of 2.0 mM −1 ) to the free enzyme and the binary enzyme · oxaloacetate complex, whereas propionyl‐CoA inhibits mainly through the formation of a ternary enzyme · propionyl‐CoA · oxaloacetate complex with a stability constant of 150 mM −1 . 3. The affinity of citrate synthase for acetyl‐CoA and propionyl‐CoA increases about 20‐fold on the binding of oxaloacetate to the enzyme. This enhancement of the binding‐strength is suggested to be due to an oxaloacetate‐induced interaction between enzyme or enzyme‐bound oxaloacetate and the acyl moiety of acetyl‐CoA and propionyl‐CoA, which has the function of accommodating the acyl group properly in the active site.