Long-range allosteric transitions in carbamoyl phosphate synthetase

James B. Thoden, Xinyi Huang, Jungwook Kim, Frank M. Raushel, Hazel M. Holden

Research output: Contribution to journalArticle

15 Citations (Scopus)

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 ∼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.

Original languageEnglish (US)
Pages (from-to)2398-2405
Number of pages8
JournalProtein Science
Volume13
Issue number9
DOIs
StatePublished - Sep 2004
Externally publishedYes

Fingerprint

Carbamyl Phosphate
Ligases
Uridine Monophosphate
Glutamine
Molecules
Mutant Proteins
Escherichia coli
Catalytic Domain
Enzymes
Adenosine Triphosphate
Glutaminase
Inosine Monophosphate
Ornithine
Biosynthesis
Bicarbonates
Ports and harbors
Crystallization
Ammonia
Aspartic Acid
Purification

Keywords

  • Amidotransferase
  • Arginine biosynthesis
  • Nucleotide binding
  • Protein structure
  • Pyrimidine biosynthesis
  • Substrate channeling
  • X-ray crystallography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Thoden, J. B., Huang, X., Kim, J., Raushel, F. M., & Holden, H. M. (2004). Long-range allosteric transitions in carbamoyl phosphate synthetase. Protein Science, 13(9), 2398-2405. https://doi.org/10.1110/ps.04822704

Long-range allosteric transitions in carbamoyl phosphate synthetase. / Thoden, James B.; Huang, Xinyi; Kim, Jungwook; Raushel, Frank M.; Holden, Hazel M.

In: Protein Science, Vol. 13, No. 9, 09.2004, p. 2398-2405.

Research output: Contribution to journalArticle

Thoden, JB, Huang, X, Kim, J, Raushel, FM & Holden, HM 2004, 'Long-range allosteric transitions in carbamoyl phosphate synthetase', Protein Science, vol. 13, no. 9, pp. 2398-2405. https://doi.org/10.1110/ps.04822704
Thoden JB, Huang X, Kim J, Raushel FM, Holden HM. Long-range allosteric transitions in carbamoyl phosphate synthetase. Protein Science. 2004 Sep;13(9):2398-2405. https://doi.org/10.1110/ps.04822704
Thoden, James B. ; Huang, Xinyi ; Kim, Jungwook ; Raushel, Frank M. ; Holden, Hazel M. / Long-range allosteric transitions in carbamoyl phosphate synthetase. In: Protein Science. 2004 ; Vol. 13, No. 9. pp. 2398-2405.
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