Dissection of the functional domains of Escherichia coli carbamoyl phosphate synthetase by site-directed mutagenesis.
The catalytic functions of the amino-terminal and carboxyl-terminal halves of the large subunit of carbamoyl phosphate synthetase from Escherichia coli have been identified using site-directed mutagenesis. Glycine residues at positions 176, 180, and 722 within the putative mononucleotide-binding site were replaced with isoleucine residues. Each of these mutations resulted in at least a 1 order of magnitude reduction in the Vmax for carbamoyl phosphate synthesis. The mutations on the amino-terminal half, G176I and G180I, caused slight reduction in the rate of synthesis of ATP from ADP and carbamoyl phosphate (the partial ATP synthesis reaction) but the bicarbonate-dependent ATPase reaction velocity was reduced to less than 10% of the wild-type rate. The mutant G722I, which is on the carboxy-terminal half, caused the partial ATP synthesis reaction to be reduced by 1 order of magnitude but the bicarbonate-dependent ATPase reaction was reduced only slightly. All three mutations are within regions which show homology to the putative glycine-rich loops of many ATP-binding proteins. These results have been interpreted to suggest that the two homologous halves of the large subunit of carbamoyl phosphate synthetase each contain a binding site for ATP. The NH2-terminal domain contains the portion of the large subunit that is primarily involved with the phosphorylation of bicarbonate to carboxy phosphate while the COOH-terminal domain contains the region of the enzyme that catalyzes the phosphorylation of carbamate to carbamoyl phosphate.
Department of Biochemistry, Texas A & M University, College Station 77843.
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000595:Amino Acid Sequence; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002221:Carbamyl Phosphate / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005998:Glycine; D007532:Isoleucine; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D012689:Sequence Homology, Nucleic Acid; D014169:Transformation, Bacterial
D007532:Isoleucine; D002221:Carbamyl Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D005998:Glycine