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Dissection of the functional domains and ATP binding sites of carbamoyl phosphate synthetase from Escherichia coli
The putative domains of the large subunit of carbamoyl phosphate synthetase from Escherichia coli have been isolated and characterized for carbamoyl phosphate synthesis, ATP synthesis, and bicarbonate-dependent ATPase activity. Through analysis of five different constructions of the large subunit, it has been determined that the putative domains are not independently active units within the protein. The results suggest that the entire length of the large subunit polypeptide is required for activity and stability. Additionally, at least the carboxy-terminal domain appears to be necessary for adequate small subunit interaction. The catalytic functions of the amino-terminal and carboxy-terminal halves of the large subunit of carbamoyl phosphate synthetase have been identified using site-directed mutagenesis. 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 one order of magnitude but the bicarbonate-dependent ATPase reaction was reduced only slightly. These results have been interpreted to suggest that the amino-terminal domain contains the portion of the large subunit that is involved with phosphorylation of bicarbonate to carboxyphosphate. The carboxy-terminal domain contains a region of the enzyme that catalyzes the phosphorylation of carbamate to carbmoyl phosphate. Site-directed mutagenesis was additionally used to substitute two strictly conserved histidine residues on the large subunit of carbamoyl phosphate synthetase with asparagine residues. Based on preliminary data from pH rate profiles and chemical modification studies with diethylpyrocarbonate, it is suggested that one or more histidine residues may play a role in the carbamoyl phosphate synthetase mechanism. Site-directed replacement of histidine-243 to asparagine resulted in the loss of all catalytic activity for carbamoyl phosphate synthesis, but the partial ATPase and ATP synthesis activities remained unchanged. The replacement of histidine-788 to asparagine also resulted in loss of carbamoyl phosphate synthesis. The partial ATPase activity, however, was reduced ten-fold. This activity could be restored in the presence of saturating concentrations of glutamine. The partial ATP synthetase activity was unaffected in the H788N mutant. These results indicate that the reaction of ammonia with carboxyphosphate has been disrupted by these modifications.
1990 Dissertation P851
Binding sites (Biochemistry)
Post, Laura Elaine (1990). Dissection of the functional domains and ATP binding sites of carbamoyl phosphate synthetase from Escherichia coli. Texas A&M University. Texas A&M University. Libraries. Available electronically from
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