Three catalytic domains of the Escherichia coli carbamoyl-phosphate synthetase (EC 6.3.5.5) have been identified in previous studies. These include the glutamine amide-N transfer domain in the carboxyl-terminal half of the glutaminase component and at least two adenine nucleotide binding sites in the synthetase component. To delineate the domains involved in subunit interactions, we have examined the effects of deletions and point mutations in the glutaminase and synthetase subunits on formation of the af6 holoenzyme. Deletion of the amino-terminal third of the glutaminase subunit abolishes interactions with the synthetase subunit, suggesting that this domain functions to stabilize the complex. Two subunit binding domains have been identified in the synthetase subunit. They are homologous to one another and are located in the amino-terminal and central regions of the synthetase component. These domains are adjacent to regions of the synthetase previously proposed to be involved in ATP binding and, possibly, activation of CO2. The new data enlarge the definition of the structural and functional domains in the two interdependent components of carbamoyl-phosphate synthetase.In Escherichia coli and most other bacteria, carbamoyl phosphate, an intermediate of arginine and pyrimidine biosynthesis, is synthesized by a single enzyme (1), glutaminedependent carbamoyl-phosphate synthetase [carbon dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5]. Bacterial carbamoyl-phosphate synthetase is composed of two subunits (2, 3). The smaller subunit, encoded by carA (4), cleaves glutamine and transfers the resultant NH3 to the larger synthetase subunit for carbamoyl phosphate synthesis (5). The glutaminase subunit of carbamoyl-phosphate synthetase appears to comprise two evolutionarily distinct domains (6). The carboxyl-terminal half is homologous to a large number of glutamine amidotransferases (6-8) and is most likely concerned with the transfer of glutamine amide N to the synthetase subunit. The function of the amino-terminal half of the glutaminase subunit is still conjectural. The structure of the large synthetase subunit, encoded by carB (9), is also interesting. This protein is composed oftwo homologous halves. Each half has been proposed to contain at least one composite or, possibly, two separate adenine nucleotide binding sites (9, 10) whose precise functions in catalysis and regulation are not known.As part of a broader study aimed at delineating the structural and functional domains of the glutaminase and synthetase subunits, we have undertaken a mutational analysis of the E. coli carbamoyl-phosphate synthetase. Different regions of each subunit have been deleted, and the mutant proteins have been analyzed for their abilities to form a physical complex. The results of mutational analysis have allowed us to demarcate the regions of the glutaminase and synthetase subunits critical for subunit interactions and for the expression of their respective catalytic functions.
The synthetase subunit of Escherichia coli carbamyl phosphate synthetase has two catalytic nucleotide-binding domains, one involved in the activation of HCO3- and the second in phosphorylation of carbamate. Here we show that a Glu841----Lys841 substitution in a putative ATP-binding domain located in the carboxyl half of the synthetase abolishes overall synthesis of carbamyl phosphate with either glutamine or NH3 as the nitrogen source. Measurements of partial activities indicate that while HCO3(-)-dependent ATP hydrolysis at saturating concentrations of substrate proceeds at higher than normal rates, ATP synthesis from ADP and carbamyl phosphate is nearly completely suppressed by the mutation. These results indicate Glu841 to be an essential residue for the phosphorylation of carbamate in the terminal step of the catalytic mechanism. The Lys841 substitution also affects the kinetic properties of the HCO3- activation site. Both kcat and Km for ATP increase 10-fold, while Km for HCO3- is increased 100-fold. Significantly, NH3 decreases rather than stimulates Pi release from ATP in the HCO3(-)-dependent ATPase reaction. The increase in kcat of the HCO3(-)-dependent ATPase reaction, and an impaired ability of the Lys841 enzyme to catalyze the reaction of NH3 with carboxy phosphate, strongly argues for interactions between the two catalytic ATP sites that couple the formation of enzyme-bound carbamate with its phosphorylation.
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