Long-range allosteric transitions in carbamoyl phosphate synthetase

Abstract

Carbamoyl phosphate synthetase plays a key role in both pyrimidine and arginine biosynthesis by catalyzing the production of carbamoyl phosphate from one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli consists of two polypeptide chains referred to as the small and large subunits, which contain a total of three separate active sites that are connected by an intramolecular tunnel. The small subunit harbors one of these active sites and is responsible for the hydrolysis of glutamine to glutamate and ammonia. The large subunit binds the two required molecules of MgATP and is involved in assembling the final product. Compounds such as L-ornithine, UMP, and IMP allosterically regulate the enzyme. Here, we report the three-dimensional structure of a site-directed mutant protein of carbamoyl phosphate synthetase from E. coli, where Cys 248 in the small subunit was changed to an aspartate. This residue was targeted for a structural investigation because previous studies demonstrated that the partial glutaminase activity of the C248D mutant protein was increased 40-fold relative to the wild-type enzyme, whereas the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished. Remarkably, although Cys 248 in the small subunit is located at approximately 100 A from the allosteric binding pocket in the large subunit, the electron density map clearly revealed the presence of UMP, although this ligand was never included in the purification or crystallization schemes. The manner in which UMP binds to carbamoyl phosphate synthetase is described.

Figure 1.

Ribbon representation of the CPS α,β-heterodimer. The small subunit is shown in magenta. The large subunit is color coded in green, yellow, blue, and red to indicate those regions defined by Met 1 to Glu 403, Val 404 to Ala 553, Asn 554 to Asn 936, and Ser 937 to Lys 1073, respectively. In the presence of allosteric activators such as L-ornithine, α,β-heterodimers associate to form an (α,β)₄-tetrameric species. The green and blue portions of the large subunit are referred to as the carboxy phosphate and carbamoyl phosphate domains. The tunnel connecting the three active sites is depicted in a gray chicken wire representation. The position of the C248D mutation in the small subunit is indicated.

Figure 2.

The site of mutation in the small subunit. A close-up view of the region surrounding Cys 248 in the small subunit of the wild-type protein is presented in A. X-ray coordinates used for this figure were from the Protein Data Bank (1JDB). (B) A superposition of the region near the site of the mutation for the wild-type and the C248D proteins is shown. The wild-type and mutant protein structures are depicted in yellow and blue, respectively. (C) A superposition of the polypeptide-chain traces for the wild-type and the C248D mutant proteins depicted in blue and gold, respectively.

Figure 3.

Representative electron density. The region near the site of the C248D mutation is shown in A, whereas the electron density corresponding to the bound UMP is displayed in B. The electron density maps shown were calculated with coefficients of the form (F_o − F_c) and contoured at 3σ.

Figure 4.

Electrostatic interactions between CPS and the IMP or UMP ligands. Schematic representations of the hydrogen bonding patterns between the protein and the IMP and UMP ligands are depicted in A and B, respectively.

Figure 5.

Binding of UMP to CPS. Those amino acid residues lying within ~5.0 Å of the UMP moiety are shown in A. A superposition of the UMP and IMP ligands, displayed in blue and yellow, respectively, is shown in B.

Scheme 1