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 synthesis of carbamoyl phosphate from bicarbonate, ammonia, and two molecules of MgATP. The enzyme is composed of two nonidentical subunits. The small subunit catalyzes the hydrolysis of glutamine to glutamate and ammonia. The large subunit catalyzes the formation of carbamoyl phosphate and has the binding sites for bicarbonate, ammonia, MgATP, and the allosteric ligands IMP, UMP, and ornithine. The allosteric ligands are believed to bind to the extreme C-terminal portion of the large subunit. Truncation mutants were constructed to investigate the allosteric binding domain. Stop codons were introduced at various locations along the carB gene in order to delete amino acids from the carboxy-terminal end of the large subunit. Removal of 14-119 amino acids from the carboxy-terminal end of the large subunit resulted in significant decreases 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 observed for the Delta 50, Delta 65, Delta 91, and Delta 119 mutant proteins. However, fort-nation of carbamoyl phosphate was detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects were observed for UMP with either the Delta 91 or Delta 119 truncation mutants, but alterations in the catalytic activity were observed in the presence of ornithine even after the removal of the last 119 amino acids from the large subunit of CPS. Six conserved amino acids within the allosteric domain were mutated. 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 mutated to alanine, valine, and isoleucine. The other amino acids were changed to alanine residues. The allosteric effects exhibited by both ornithine and UMP were gradually diminished as the glycine residue at position 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 carbamoyl phosphate. The T977A mutant was not regulated by UMP, but the full allosteric effects were observed with ornithine. The R1030A and R1031A mutants exhibited wild-type properties whereas the N1015A mutant could not be purified. These results demonstrate that the allosteric effects exhibited by UMP and ornithine can be functionally separated. The mutation of a single conserved threonine residue switches off the allosteric effects exhibited by UMP while not altering the ability of this protein to be activated by ornithine. In contrast, the allosteric effects exhibited by both UMP and ornithine can be gradually diminished by the substitution of a single glycine residue at position 968 with larger hydrophobic side chains.