Dissection of the functional domains of Escherichia coli carbamoyl phosphate synthetase by site-directed mutagenesis.

摘要: The catalytic functions of the amino-terminal and carboxyl-terminal halves large subunit carbamoyl phosphate synthetase from Escherichia coli have been identified using site-directed mutagenesis. Glycine residues at positions 176, 180, 722 within putative mononucleotide-binding site were replaced with isoleucine residues. Each these mutations resulted in least a 1 order magnitude reduction Vmax for synthesis. on half, G176I G180I, caused slight rate synthesis ATP ADP (the partial reaction) but bicarbonate-dependent ATPase reaction velocity was reduced to less than 10% wild-type rate. mutant G722I, which is carboxy-terminal be by only slightly. All three are regions show homology glycine-rich loops many ATP-binding proteins. These results interpreted suggest that two homologous each contain binding ATP. NH2-terminal domain contains portion primarily involved phosphorylation bicarbonate carboxy while COOH-terminal region enzyme catalyzes carbamate phosphate.