Evidence that Citrate Synthase Operates by an Ordered Ternary‐Complex Mechanism

Abstract

Gel equilibrium‐diffusion measurements show that acetyl‐CoA binds to two indistinguishable sites in citrate synthase with a dissociation constant of 0.13 mM, which exceeds Km for acetyl‐CoA by a factor of about 20. This observation is inconsistent with previous suggestions that the citrate synthase‐catalyzed condensation reaction between acetyl‐CoA and oxaloacetate proceeds by a rapid‐equilibrium random‐order mechanism, and provides strong evidence that the mechanism is effectively ordered with oxaloacetate adding first to the enzyme, which makes it possible to calculate on and off constants for the binding of oxaloacetate (k2= 4 × 109 min−1 M−1 and k−2= 2 × 104 min−1). Confirmatory evidence for this interpretation of the experimental data is given by the product‐inhibition pattern observed in the presence of citrate. Copyright © 1974, Wiley Blackwell. All rights reserved