The differentially conserved residues of carbamoyl-phosphate synthetase

Carbamoyl-phosphate synthetase (CPS) from Escherichia coli is a heterodimer ic protein. The larger of the two subunits (M-r similar to 118,000) contain s a pair of homologous domains of approximately 400 residues each that are similar to 40% identical in amino acid sequence. The carboxy phosphate (res idues 1-400) and carbamoyl phosphate domains (residues 553-933) also contai n similar to 79 differentially conserved residues. These are residues that are conserved throughout the bacterial evolution of CPS in one of these hom ologous domains but not the other. The role of these differentially conserv ed residues in the structural and catalytic properties of CPS was addressed by swapping segments of these residues from one domain to the other. Nine of these chimeric mutant enzymes were constructed, expressed, purified, and characterized. A majority of the mutants were unable to synthesize any car bamoyl phosphate and the rest were severely crippled. True tandem repeat ch imeric proteins were constructed by the complete substitution of one homolo gous domain sequence for the other. Neither of the two possible chimeric pr oteins was structurally stable. These results have been interpreted to demo nstrate that the two homologous domains in the large subunit of CPS are fun ctionally and structurally nonequivalent. This nonequivalence is a direct r esult of the specific functions each of these domains must perform during t he overall synthesis of carbamoyl phosphate in the wild type enzyme and the specific structural alterations imposed by the differentially conserved re sidues.