Escherichia coli carbamoyl-phosphate synthetase: domains of glutaminase and synthetase subunit interaction.

Abstract

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.

Specificity Determining Residues in Ammonia- and Glutamine-dependent Carbamoyl Phosphate Synthetases

“…Carbamoyl phosphate synthetases (CPSs) are made up of a two-domain glutamine amidotransferase (GAT) component and a four-domain synthetase (SYN) component (1)(2)(3)(4)(5). The N-terminal domain of the GAT component is required for interaction with the SYN component (6,7), and the C-terminal domain has a triad-type GAT structure (5,8). In glutamine-utilizing CPSs (e.g.…”

“…The ammonia thus liberated is subsequently channeled to the SYN component where it, together with bicarbonate, is used to generate CP with an energy expenditure of two ATP molecules per CP synthesized. The SYN component consists of two ATPutilizing domains (13), one allosteric interaction domain (14 -17), and one domain for interaction with the GAT component (6,7). In the absence of other CPS substrates, the GAT domain is able to catalyze a glutaminase reaction, and in the absence of glutamine, the SYN component is able to synthesize CP from NH 3 (18 -20).…”

Unmasking a Functional Allosteric Domain in an Allosterically Nonresponsive Carbamoyl-phosphate Synthetase

“…Domains B and C (403 and 382 residues, respectively) are regions of internal duplication (31) and each domain contains one ATP binding site (29,30). The 149-residue domain DЈ is involved in oligomerization (29,30,32). Domain D, the 136-residue C-terminal region of the eCPS synthetase subunit, termed the "allosteric domain" (29,30), is critical for CP synthesis (13), and is also involved in oligomerization (29,30).…”

Effect of growth temperature on folding of carbamoylphosphate synthetases of Salmonella typhimurium and a cold-sensitive derivative

“…The ammonia-dependent rate is a function of the large subunit alone, whereas the glutamine-dependent rate is a function of the holoenzyme (3). Previous work (3,16,30) provided evidence for strong interactions between the large and small subunits, and the ratio obtained with the two substrates must depend on the conformation of the holoenzyme. Hill plots of the data; SO.s and n (Hill coefficient) were calculated by nonlinear regression analysis.…”