The use of nucleotide analogs to evaluate the mechanism of the heterotropic response of Escherichia coli aspartate transcarbamoylase

As an alternative method to study the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase, a series of nucleotide analogs were used . These nucleotide analogs have the advantage over site-specific mutagenesi s experiments in that interactions between the backbone of the protein and the nucleotide could be evaluated in terms of their importance for function . The ATP analogs purine 5'-triphosphate (PTP), 6-chloropurine 5'-triphosph ate (Cl-PTP), 6-mercapropurine 5'-triphosphate (SH-PTP), 6-methylpurine 5'- triphosphate (Me-PTP), and 1-methyladenosins 5'-triphosphate (Me-ATP) were partially synthesized from their corresponding nucleosides. Kinetic analysi s was performed on the wild-type enzyme in the presence of these ATP analog s along with GTP, ITP, and XTP. PTP, Cl-PTP, and SH-PTP each activate the e nzyme at subsaturating concentrations of L-aspartate and saturating concent rations of carbamoyl phosphate, but not to the same extent as does ATP. The se experiments suggest that the interaction between N-6-amino group of ATP and the backbone of the regulatory chain is important for orienting the nuc leotide and inducing the displacements of the regulatory chain backbone nec essary for initiation of the regulatory response. Me-PTP and Me-ATP also ac tivate the enzyme, but in a more complex fashion, which suggests differenti al binding at the two sites within each regulatory dimer. The purine nucleo tides GTP, ITP, and XTP each inhibit the enzyme but to a lesser extent than CTP. The influence of deoxy and dideoxynucleotides on the activity of the enzyme was also investigated. These experiments suggest that the 2' and 3' ribose hydroxyl groups are not of significant importance for binding and or ientation of the nucleotide in the regulatory binding site. 2'-dCTP inhibit s the enzyme to the same extent as CTP, indicating that the interactions of the enzyme to the O-2-carbonyl of CTP are critical for CTP binding, inhibi tion, and the ability of the enzyme to discriminate between ATP and CTP. Ex amination of the electrostatic surface potential of the nucleotides and the regulatory chain suggest that the complimentary electrostatic interactions between the nucleotides and the regulatory chain are important for binding and orientation of the nucleotide necessary to induce the local conformati onal changes that propagate the heterotropic effect.