Open AccessEvidence that Citrate Synthase Operates by an Ordered Ternary‐Complex Mechanism
Author(s) -
Johansson CarlJohan,
Pettersson Gösta
Publication year - 1974
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1974.tb03350.x
Subject(s) - citrate synthase , dissociation constant , chemistry , ternary operation , ternary complex , atp synthase , atp citrate lyase , dissociation (chemistry) , thermodynamics , enzyme , biochemistry , physics , receptor , computer science , programming language
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 K m 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 ( k 2 = 4 × 10 9 min −1 M −1 and k −2 = 2 × 10 4 min −1 ). Confirmatory evidence for this interpretation of the experimental data is given by the product‐inhibition pattern observed in the presence of citrate.