The N-terminus of the regulatory chain of Escherichia coli aspartate transcarbamoylase is important for both nucleotide binding and heterotropic effects.

X-ray crystallographic studies indicate that the N-terminal region of the regulatory chain in Escherichia coli aspartate transcarbamoylase resides close to the effector binding site. The proximity of the N-terminal region to the binding site suggests it may be important for nucleotide binding and, therefore, the heterotropic mechanism. The N-terminal region of the structure is not well-defined since the electron density in this region is weak, indicating a flexible and mobile region. Furthermore, alanine scanning mutagenesis of residues 2-7 indicated that the N-terminal region may be involved in nucleotide binding and the heterotropic mechanism, especially, UTP recognition [Dembowski, N., and Kantrowitz, E. R. (1994) Protein Eng. 7, 673-679]. In order to investigate further the role of the N-terminal region in the heterotropic mechanism, the first 10 N-terminal residues of the regulatory chain were deleted using site-specific mutagenesis. This mutant enzyme was compared to the wild-type enzyme, and both solubility and functional differences were observed. The mutant enzyme forms an insoluble aggregate which can be solubilized by the addition of nucleotides, such as CTP, suggesting that the exposed nucleotide binding site is involved in aggregate formation. Kinetic analyses of the mutant enzyme showed a lower maximal velocity and slightly lower aspartate affinity. Apparent binding constants determined for CTP, ATP, UTP, and CTP in the presence of UTP suggest the heterotropic response is also altered. This study suggests that the N-terminal region of the regulatory subunit is important for controlling nucleotide binding, creating the high-affinity and low-affinity effector binding sites, and coupling the binding sites within the regulatory dimer.