Structural defects within the carbamate tunnel of carbamoyl phosphate synthetase

Jungwook Kim, Stanley Howell, Xinyi Huang, Frank M. Raushel

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

The X-ray crystal structure of carbamoyl phosphate synthetase (CPS) from Escherichia coli has unveiled the existence of two molecular tunnels within the heterodimeric enzyme. These two interdomain tunnels connect the three distinct active sites within this remarkably complex protein and apparently function as conduits for the transport of unstable reaction intermediates between successive active sites. The operational significance of the ammonia tunnel for the migration of NH3 is supported experimentally by isotope competition and protein modification. The passage of carbamate through the carbamate tunnel has now been assessed by the insertion of site-directed structural blockages within this tunnel. Gln-22, Ala-23, and Gly-575 from the large subunit of CPS were substituted by mutagenesis with bulkier amino acids in an attempt to obstruct and/or hinder the passage of the unstable intermediate through the carbamate tunnel. The structurally modified proteins G575L, A23L/G575S, and A23L/G575L exhibited a substantially reduced rate of carbamoyl phosphate synthesis, but the rate of ATP turnover and glutamine hydrolysis was not significantly altered. These data are consistent with a model for the catalytic mechanism of CPS that requires the diffusion of carbamate through the interior of the enzyme from the site of synthesis within the N-terminal domain of the large subunit to the site of phosphorylation within the C-terminal domain. The partial reactions of CPS have not been significantly impaired by these mutations, and thus, the catalytic machinery at the individual active sites has not been functionally perturbed.

Original languageEnglish (US)
Pages (from-to)12575-12581
Number of pages7
JournalBiochemistry
Volume41
Issue number42
DOIs
StatePublished - Oct 22 2002
Externally publishedYes

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Carbamyl Phosphate
Carbamates
Ligases
Tunnels
Defects
Catalytic Domain
Proteins
Enzymes
Reaction intermediates
Mutagenesis
Phosphorylation
Glutamine
Ammonia
Isotopes
Hydrolysis
Escherichia coli
Adenosine Triphosphate
Machinery
X-Rays
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural defects within the carbamate tunnel of carbamoyl phosphate synthetase. / Kim, Jungwook; Howell, Stanley; Huang, Xinyi; Raushel, Frank M.

In: Biochemistry, Vol. 41, No. 42, 22.10.2002, p. 12575-12581.

Research output: Contribution to journalArticle

Kim, J, Howell, S, Huang, X & Raushel, FM 2002, 'Structural defects within the carbamate tunnel of carbamoyl phosphate synthetase', Biochemistry, vol. 41, no. 42, pp. 12575-12581. https://doi.org/10.1021/bi020421o
Kim, Jungwook ; Howell, Stanley ; Huang, Xinyi ; Raushel, Frank M. / Structural defects within the carbamate tunnel of carbamoyl phosphate synthetase. In: Biochemistry. 2002 ; Vol. 41, No. 42. pp. 12575-12581.
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