An aspartate transcarbamylase lacking catalytic subunit interactions. II. Regulatory subunits are responsible for the lack of co-operative interactions between catalytic sites. Drastic feedback inhibition does not restore these interactions

It has been previously reported (Kerbiriou &; Hervé, 1972) that, when a uracil-requiring mutant of Escherichia coli is derepressed for the biosynthesis of the enzymes of the pyrimidine pathway in the presence of 2-thiouracil, it synthesizes a modified aspartate transcarbamylase which is still sensitive to the feedback inhibitor CTP, but which does not show homotropic positive interactions between catalytic sites. It is shown here that these homotropic interactions do not reappear upon strong inhibition by CTP, indicating that the two types of interactions are really disconnected and must involve different molecular mechanisms. CTP is acting at the level of the apparent K_m of the enzyme for aspartate. It is also the case for ATP, which stimulates 2-thiouracil aspartate-transcarbamylase. Kinetic studies of the hybrid molecules made up of subunits prepared from normal and modified enzymes show that it is a modification at the level of the regulatory subunits which is responsible for the lack of co-operative interactions between catalytic sites. These results are discussed in terms of a four-state model.