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Long-range allosteric transitions in carbamoyl phosphate synthetase

¹ Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA
² Department of Chemistry, Texas A&M University, College Station, Texas 77843, USA

(RECEIVED April 20, 2004; FINAL REVISION May 24, 2004; ACCEPTED May 24, 2004)

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 ~100 Å 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.

Keywords: X-ray crystallography; protein structure; nucleotide binding; substrate channeling; amidotransferase; pyrimidine biosynthesis; arginine biosynthesis

Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04822704.

Reprint requests to: Hazel M. Holden, Department of Biochemistry, University of Wisconsin, Madison, WI, 53706, USA; e-mail: Hazel_Holden{at}biochem.wisc.edu; fax: (608) 262-1319; or Frank M. Raushel, Department of Chemistry, Texas A&M University, College Station, TX 77843, USA; e-mail: Raushel{at}tamu.edu; fax: (979) 845-9452.

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