Carbamate transport in carbamoyl phosphate synthetase: a theoretical and experimental investigation.

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 traveling nearly 40 A 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.

A combined theoretical and experimental study of the ammonia tunnel in carbamoyl phosphate synthetase.

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 approximately 45 A 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.

Mechanism for the transport of ammonia within carbamoyl phosphate synthetase determined by molecular dynamics simulations.

Carbamoyl phosphate synthetase (CPS) is a member of the amidotransferase family of enzymes that uses the hydrolysis of glutamine as a localized source of ammonia for biosynthetic transformations. Molecular dynamics simulations for the transfer of ammonia and ammonium through a tunnel in the small subunit of CPS resulted in five successful trajectories for ammonia transfer, while ammonium was immobilized in a water pocket inside the small subunit of the heterodimeric protein. The observed molecular tunnel for ammonia transport is consistent with that suggested by earlier X-ray crystallography and site-directed mutation studies.

Tunneling of intermediates in enzyme-catalyzed reactions.

A fascinating group of enzymes has been shown to possess multiple active sites connected by intramolecular tunnels for the passage of reactive intermediates from the site of production to the site of utilization. In most of the examples studied to date, the binding of substrates at one active site enhances the formation of a reaction intermediate at an adjacent active site. The most common intermediate is ammonia, derived from the hydrolysis of glutamine, but molecular tunnels for the passage of indole, carbon monoxide, acetaldehyde and carbamate have also been identified.