A Combined Theoretical and Experimental Study of the Ammonia Tunnel in Carbamoyl Phosphate Synthetase

Abstract

The transfer of ammonia in carbamoyl phosphate synthetase (CPS) was investigated by molecular dynamics simulations and experimental characterization of mutations within the ammonia tunnel. In CPS, ammonia is derived from the hydrolysis of glutamine and this intermediate must travel ∼45 Å from the site of formation in the small subunit to the site of utilization in the large subunit. In this investigation the migration of ammonia was analyzed from the exit of the small subunit through the large subunit where it ultimately reacts with the carboxy phosphate intermediate. Potential of mean force calculations along the transfer pathway for ammonia indicate a relatively low free energy barrier for the translocation of ammonia. The highest barrier of 7.2 kcal/mol is found at a narrow turning gate surrounded by the side chains of Cys-232, Ala-251 and Ala-314 in the large subunit. The environment of the ammonia tunnel from the exit of the small subunit to the turning gate in the tunnel is filled with clusters of water molecules and the ammonia is able to travel through this area easily. After ammonia passes through the turning gate it enters a hydrophobic passage. A hydrogen bond then forms between the ammonia and Thr-249, which facilitates the delivery to a more hydrophilic environment near the active site for the reaction with the carboxy phosphate intermediate. The transport process from the turning gate to the end of the tunnel is favored by an overall down-hill free energy potential and no free energy barrier higher than 3 kcal/mol. A conformational change of the turning gate, caused by formation of the carboxy phosphate intermediate, is consistent with a mechanism in which the reaction between ATP and bicarbonate triggers the transport of ammonia and consequently accelerates the rate of glutamine hydrolysis in the small subunit. A blockage in the turning gate passageway was introduced by the triple mutant C232V/A251V/A314V. This mutant is unable to synthesize carbamoyl phosphate using glutamine as a nitrogen source.

Gates of Enzymes

“…32−34 This methodology was used to compare the results of computational and experimental studies on the passage of the tetramethylammonium cation through acetylcholinesterase 35 and migration of ammonia through carbamoyl phosphate synthetase. 36 …”

“…Gates of this kind can therefore switch between states at different frequencies, which can be adjusted by mutating the gating residues. Aperture-type gates have been identified in several enzymes including carbamoyl phosphate synthetase, 36 choline oxidase, 31 glutamate synthases, 62 extradiol dioxygenases-homoprotocatechuate 2,3-dioxygenase, 63 cytochrome P450 eryF , 25 and acetylcholinesterase. 64 …”

Fluorescent protein barrel fluctuations and oxygen diffusion pathways in mCherry

“…25 Recent developments in efficient computational sampling methods have allowed thorough scanning of the possible pathways for gas diffusion in the interior of proteins. [26][27][28][29] For example, such computational investigations have proved useful in understanding gas diffusion in many protein systems such as molecular dioxygen pathways via dynamic oxygen access channels in flavoproteins, [30][31][32] ammonia transport in carbamoyl phosphate synthetase, [33][34][35] and gas diffusion and channeling in hemoglobin. 28,36 In this paper, we use explicit solvent all-atom molecular dynamics (MD) simulations to investigate the protein barrel fluctuations in mCherry, which is one of the most useful monomeric variants of RFP.…”

Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol

“…Passage of an intermediate directly from one active site to another without equilibration with the solvent is called channeling (26)(27)(28)(29)(30). Examples of reactive intermediates that are channeled from one active site to another include carbamate (31), ammonia (32), indole (33), aspartyl phosphate (34), glutamyl phosphate (35), and phosphoribosylamine (26). TCBQ is even more reactive than these intermediates, undergoing reaction with thiols at physiologically relevant concentrations in milliseconds.…”