Eukaryotes have two forms of carbamoyl phosphate synthetase (CPS), while E.coli

Question

Eukaryotes have two forms of carbamoyl phosphate synthetase (CPS), while E.coli have only one form. Discuss how the E.coli CPS is regulated with regard to the urea cycle. The text states that CPS can produce carbamoyl phosphate even when the glutaminase activity is eliminated. Explain why this is so. The active sites for the syntheses of carboxyphosphate, ammonia, and carbamoyl phosphate are physically separated from one another. How is the location of these three sites proposed to contribute to product formation? Is this an efficient mechanism? Why or why not?

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Explanation / Answer

Carbamoyl phosphate synthetase from E. coli catalyzes the first committed step in the separate biosynthetic pathways for the production of arginine, and pyrimidine nucleotides. The enzyme is an ?,?-heterodimer consisting of a small amidotransferase subunit complexed to a larger synthetase subunit. The small subunit, encoded by carA gene hydrolyzes glutamine to glutamate and ammonia. The large subunit, encoded by carB gene binds the two required molecules of MgATP and catalyzes the two required phosphorylation events. The small subunit contains a catalytic triad (Cys269, His353 and Glu355) situated between the two structural domains. The large subunit consists of four structural units: the carboxyphosphate synthetic component, the oligomerization domain, the carbamoyl phosphate synthetic component and the allosteric domain.

The enzyme contains a total of three separate active sites that are connected by an intramolecular tunnel. The small subunit harbors one of these active sites. Two molecular tunnels connect the three active sites contained within the small and large subunits. The ammonia tunnel extends from the active site of the small subunit to the active site of the carboxy phosphate domain of the large subunit. The carbamate tunnel connects the two active sites within the large subunit.

The active sites of the enzyme are very resistant toward changes in nucleotide specificity, most likely owing to the presence of the K+-binding loop that prevents binding of GTP. Mutations at positions 144 and 690 interfere with access of substrate to the active sites.

Site directed mutagenesis showed that either a C269G or C269S mutation results in loss of all of the glutamine-dependent synthesis of carbamoylphosphate though the enzyme still retains the ability to use ammonia as the nitrogen sourc.
Mutagenesis of residues Glu783, Glu892 and Thr1042 that had been shown by crystallography to interact with bound ornithine demonstrated their importance for ornithine binding and provided insight into the mechanism of allosteric control of enzyme activity . Site-directed mutagenesis studies also suggested that Cys269 and His353 of the conserved catalytic triad in the glutamine aminotransferase site may function as a catalytic dyad.

The transfer of ammonia through the ammonia tunnel in this enzyme was investigated using a combination of molecular dynamics simulations and experimental characterization of mutations. This approach was also used to characterize the transport of carbamate through the large subunit of this enzyme .

In another mutagenesis study, substiturion of six intrinsic tryptophan residues in the enzyme with tyrosine created a tryptophan-free variant. Subsequent substitutions of individual tryptophan residues at each site allowed their use as fluoresence probes to study conformational changes.

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