Heterogeneity of sites in isolated catalytic subunits of aspartate transcarbamoylase.

Carbamoyl phosphate, a substrate of aspartate transcarbamoylase from Escherichia coli, binds with different modes of association in 3 sites in the unmodified catalytic subunits. Over a narrow pH range (6.6--8.0), positive, negative or no interactions are observed. Several substrate analogues also bind to 3 sites in the catalytic trimer. The association of pyridoxal phosphate, CTP and ATP, all competitive inhibitors of carbamoyl phosphate, exhibit negative interactions. Binding of succinate, an analogue of the second substrate, aspartate, is also characterized by heterogeneity. Dissociation constants to high and low-affinity sites differ by factors of 10-100. These observations clearly indicate that, although not observed kinetically, the active sites in the catalytic subunits of aspartate transcarbamoylase are heterogenous.