Perforation of the Tunnel Wall in Carbamoyl Phosphate Synthetase Derails the Passage of Ammonia between Sequential Active Sites

Jungwook Kim, Frank M. Raushel

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Carbamoyl phosphate synthetase (CPS) from Escherichia coli consists of a small subunit (∼42 kDa) and a large subunit (∼118 kDa) and catalyzes the biosynthesis of carbamoyl phosphate from MgATP, bicarbonate, and glutamine. The enzyme is able to utilize external ammonia as an alternative nitrogen source when glutamine is absent. CPS contains an internal molecular tunnel, which has been proposed to facilitate the translocation of reaction intermediates from one active site to another. Ammonia, the product from the hydrolysis of glutamine in the small subunit, is apparently transported to the next active site in the large subunit of CPS over a distance of about 45 Å. The ammonia tunnel that connects these two active sites provides a direct path for the guided diffusion of ammonia and protection from protonation. Molecular damage to the ammonia tunnel was conducted in an attempt to induce leakage of ammonia directly to the protein exterior by the creation of a perforation in the tunnel wall. A hole in the tunnel wall was made by mutation of integral amino acid residues with alanine residues. The triple mutant αP360A/αH361A/βR265A was unable to utilize glutamine for the synthesis of carbamoyl phosphate. However, the mutant enzyme retained full catalytic activity when external ammonia was used as the nitrogen source. The synchronization of the partial reactions occurring at the three active sites observed with the wild-type CPS was seriously disrupted with the mutant enzyme when glutamine was used as a nitrogen source. Overall, the catalytic constants of the mutant were consistent with the model where the channeling of ammonia has been disrupted due to the leakage from the ammonia tunnel to the protein exterior.

Original languageEnglish (US)
Pages (from-to)5334-5340
Number of pages7
JournalBiochemistry
Volume43
Issue number18
DOIs
StatePublished - May 11 2004
Externally publishedYes

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Carbamyl Phosphate
Ligases
Ammonia
Catalytic Domain
Tunnels
Glutamine
Nitrogen
Enzymes
Reaction intermediates
Protonation
Biosynthesis
Bicarbonates
Alanine
Escherichia coli
Hydrolysis
Catalyst activity
Synchronization
Proteins
Adenosine Triphosphate
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Perforation of the Tunnel Wall in Carbamoyl Phosphate Synthetase Derails the Passage of Ammonia between Sequential Active Sites. / Kim, Jungwook; Raushel, Frank M.

In: Biochemistry, Vol. 43, No. 18, 11.05.2004, p. 5334-5340.

Research output: Contribution to journalArticle

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abstract = "Carbamoyl phosphate synthetase (CPS) from Escherichia coli consists of a small subunit (∼42 kDa) and a large subunit (∼118 kDa) and catalyzes the biosynthesis of carbamoyl phosphate from MgATP, bicarbonate, and glutamine. The enzyme is able to utilize external ammonia as an alternative nitrogen source when glutamine is absent. CPS contains an internal molecular tunnel, which has been proposed to facilitate the translocation of reaction intermediates from one active site to another. Ammonia, the product from the hydrolysis of glutamine in the small subunit, is apparently transported to the next active site in the large subunit of CPS over a distance of about 45 {\AA}. The ammonia tunnel that connects these two active sites provides a direct path for the guided diffusion of ammonia and protection from protonation. Molecular damage to the ammonia tunnel was conducted in an attempt to induce leakage of ammonia directly to the protein exterior by the creation of a perforation in the tunnel wall. A hole in the tunnel wall was made by mutation of integral amino acid residues with alanine residues. The triple mutant αP360A/αH361A/βR265A was unable to utilize glutamine for the synthesis of carbamoyl phosphate. However, the mutant enzyme retained full catalytic activity when external ammonia was used as the nitrogen source. The synchronization of the partial reactions occurring at the three active sites observed with the wild-type CPS was seriously disrupted with the mutant enzyme when glutamine was used as a nitrogen source. Overall, the catalytic constants of the mutant were consistent with the model where the channeling of ammonia has been disrupted due to the leakage from the ammonia tunnel to the protein exterior.",
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