Carbamate Transport in Carbamoyl Phosphate Synthetase: A Theoretical and Experimental Investigation

Abstract

The transport of carbamate through the large subunit of carbamoyl phosphate synthetase (CPS) from Escherichia coli was investigated by molecular dynamics and site-directed mutagenesis. Carbamate, the product of the reaction involving ATP, bicarbonate and ammonia, must be delivered from the site of formation to the site of utilization by travelling nearly 40 Å within the enzyme. Potentials of mean force (PMF) calculations along the entire tunnel for the translocation of carbamate indicate that the tunnel is composed of three continuous water pockets and two narrow connecting parts, near Ala-23 and Gly-575. The two narrow parts render two free energy barriers of 6.7 and 8.4 kcal/mol. Three water pockets were filled with about 21, 9 and 9 waters, respectively, and the corresponding relative free energies of carbamate residing in these free energy minima are 5.8, 0 and 1.6 kcal/mol, respectively. The release of phosphate into solution at the site for the formation of carbamate allows the side chain of Arg-306 to rotate towards Glu-25, Glu-383, and Glu-604. This rotation is virtually prohibited by a barrier of at least 23 kcal/mol when phosphate remains bound. This conformational change not only opens the entrance of the tunnel but also shields the charge-charge repulsion from the three glutamate residues when carbamate passes through the tunnel. Two mutants, A23F and G575F, were designed to block the migration of carbamate through the narrowest parts of the carbamate tunnel. The mutants retained only 1.7% and 3.8% of the catalytic activity for the synthesis of carbamoyl phosphate relative to the wild type CPS, respectively.

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.…”

Tunnels modulate ligand flux in a heme nitric oxide/oxygen binding (H-NOX) domain

“…For example, channels for gas transport have been identified in enzymes utilizing O 2 (e.g., oxidases and oxygenases) (1,2), N 2 (nitrogenases) (3), and H 2 (hydrogenases) (4) for critical biological redox reactions. In enzymes with multiple active sites, tunnel networks shunt oftentimes volatile or reactive intermediates between reactive centers for processes such as bacterial carbon fixation (5) and the biosynthesis of essential cofactors (6) and amino acids (7,8).…”

Structure of human carbamoyl phosphate synthetase: deciphering the on/off switch of human ureagenesis

“…At each end of the tunnel Arg721 and Arg1262 can participate, as indicated before, in the binding to the nucleotide γ-phosphate in domains L1 and L3, respectively. These arginine residues, together with five conserved glutamic acid residues at the beginning (glutamates 440, 797 and 1018) and at the end (glutamates 991 and 1334) of the tunnel, can play a role in the entry and exit of carbamate to and from the tunnel 43 44 45 .…”