Binding of alpha-ketoisovalerate to alpha-isopropylmalate synthase. Half-of-the-sites and all-of-the-sites availability.

Binding of alpha-ketoisovalerate to alpha-isopropylmalate synthase (3-hydroxy-4-methyl-3-carboxyvalerate 2-oxo-3-methylbutyrate-lyase (CoA-acetylating), EC 4.1.3.12) from Salmonella thyphimurium has been studied by equilibrium dialysis. When alpha-ketoisovalerate is the only ligand present, no more than two sites per enzyme tetramer can be saturated under the conditions chosen. The binding is non-cooperative with a dissociation constant of 6.6+/- 0.4 muM. Binding of alpha-ketoisovalerate has also been studied in the presence of propionyl-CoA. This compound was selected because of its close similarity to the natural substrate acetyl-CoA. It is a competitive inhibitor with respect to acetyl-CoA while reacting only extremely sluggishly as as substrate itself. The presence of propionyl-CoA has a profound effect on alpha-ketoisovalerate binding. The number of sites available to alpha-ketoisovalerate increases to about four per tetramer. At the same time, the dissociation constant for alpha-ketoisovalerate increases approx. 4-fold. These results suggest that the active conformation of alpha-isopropylmalate synthase is not obtained unless both substrates are present. They also support the notion, based on previous studies with the feedback inhibitor L-leucine, that alpha-isopropylmalate synthase has a tendency to form "functional dimers".