REGULATORY CHANGES IN THE CONTROL OF CARBAMOYL-PHOSPHATE SYNTHETASE INDUCED BY TRUNCATION AND MUTAGENESIS OF THE ALLOSTERIC BINDING DOMAIN

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the syn thesis of carbamoyl phosphate from bicarbonate, ammonia, and two molec ules of MgATP. The enzyme is composed of two nonidentical subunits. Th e small subunit catalyzes the hydrolysis of glutamine to glutamate and ammonia. The large subunit catalyzes the formation of carbamoyl phosp hate and has the binding sites for bicarbonate, ammonia, MgATP, and th e allosteric ligands IMP, UMP, and ornithine. The allosteric ligands a re believed to bind to the extreme C-terminal portion of the large sub unit. Truncation mutants were constructed to investigate the allosteri c binding domain. Stop codons were introduced at various locations alo ng the carB gene in order to delete amino acids from the carboxy-termi nal end of the large subunit. Removal of 14-119 amino acids from the c arboxy-terminal end of the large subunit resulted in significant decre ases in all of the enzymatic activities catalyzed by the enzyme. A 40- fold decrease in the glutamine-dependent ATPase activity was observed for the Delta 14 truncation. Similar losses in activity were also obse rved for the Delta 50, Delta 65, Delta 91, and Delta 119 mutant protei ns. However, fort-nation of carbamoyl phosphate was detected even afte r the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects were observed for UMP with eithe r the Delta 91 or Delta 119 truncation mutants, but alterations in the catalytic activity were observed in the presence of ornithine even af ter the removal of the last 119 amino acids from the large subunit of CPS. Six conserved amino acids within the allosteric domain were mutat ed. These sites included two glycine residues at positions 921 and 968 , a threonine at position 977, an asparagine at position 1015, and two arginines at positions 1030 and 1031. The glycine residues were mutat ed to alanine, valine, and isoleucine. The other amino acids were chan ged to alanine residues. The allosteric effects exhibited by both orni thine and UMP were gradually diminished as the glycine residue at posi tion 968 was changed to alanine, valine, and finally isoleucine. This effect was observed in both the glutamine-dependent ATP hydrolysis and the ATP synthesis reactions. The G921A mutant showed no alteration in any of the allosteric properties. The mutant proteins G921V and C921I were unstable and were found to be defective for the synthesis of car bamoyl phosphate. The T977A mutant was not regulated by UMP, but the f ull allosteric effects were observed with ornithine. The R1030A and R1 031A mutants exhibited wild-type properties whereas the N1015A mutant could not be purified. These results demonstrate that the allosteric e ffects exhibited by UMP and ornithine can be functionally separated. T he mutation of a single conserved threonine residue switches off the a llosteric effects exhibited by UMP while not altering the ability of t his protein to be activated by ornithine. In contrast, the allosteric effects exhibited by both UMP and ornithine can be gradually diminishe d by the substitution of a single glycine residue at position 968 with larger hydrophobic side chains.