PMID:2186028
| Citation |
Post, LE, Post, DJ and Raushel, FM (1990) Dissection of the functional domains of Escherichia coli carbamoyl phosphate synthetase by site-directed mutagenesis. J. Biol. Chem. 265:7742-7 |
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| Abstract |
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. |
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| Keywords |
Adenosine Diphosphate/metabolism; Adenosine Triphosphate/biosynthesis; Amino Acid Sequence; Binding Sites; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/isolation & purification; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/metabolism; Carbamyl Phosphate/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Glycine; Isoleucine; Kinetics; Molecular Sequence Data; Mutation; Sequence Homology, Nucleic Acid; Transformation, Bacterial |
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