Substrate-inhibiton by acetyl-CoA in the condensation reaction between oxaloacetate and acetyl-CoA catalyzed by citrate synthase from pig heart.

Deviations from Michealis-Menten kinetics in the pig-heart citrate synthase (citrate-oxaloacetate-lyase(pro-3S-CH2-COO-leads to acetyl-CoA), EC 4.1.3.7) system have been characterized and analyzed in view of the kinetic theory described in the preceding paper. The enzymic condensation reaction between acetyl-CoA and oxaloacetate is subject to substrate-inhibition by acetyl-CoA. This can be attributed to the formation of a productive enzyme-acetyl-CoA complex with a dissociation constant of 110 uM. The binding of acetyl-CoA to the enzyme decreases the on-velocity constant for oxaloacetate-binding from 4000 min-1- micrometer-1 to 1700 min-1-micrometer-1. The affinity of citrate synthase for oxaloacetate increase at least 20-fold on the binding of acetyl-CoA. The latter cooperativity effect can be attributed to a more than 45-fold decrease of the off-velocity constant for oxaloacetate-binding.