Literature summary for 6.3.5.5 extracted from

Lund, L.; Fan, Y.; Shao, Q.; Gao, Y.; Raushel, F.
Carbamate transport in carbamoyl phosphate synthetase: A theoretical and experimental investigation (2010), J. Am. Chem. Soc., 132, 3870-3878.

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
molecular dynamics simulation. 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. The tunnel is composed of three continuous water pockets and two narrow connecting parts, near residues A23 and G575. The two narrow parts render two free energy barriers of 6.7 and 8.4 kcal/mol, respectively. Three water pockets are 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 R306 to rotate toward E25, E383, and E604. 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	Escherichia coli

Crystallization (Comment)

Organism

molecular dynamics simulation. 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. The tunnel is composed of three continuous water pockets and two narrow connecting parts, near residues A23 and G575. The two narrow parts render two free energy barriers of 6.7 and 8.4 kcal/mol, respectively. Three water pockets are 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 R306 to rotate toward E25, E383, and E604. 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

Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
A23F	migration of carbamate through the narrowest part of the carbamate tunnel is blocked. From the kinetic data the only reaction significantly affected by this mutation is the overall synthesis of carbamoyl phosphate (only 1.7% compared to wild-type)	Escherichia coli
A23F	mutant designed to block the migration of carbamate through the narrowest parts of the carbamate tunnel. Mutant retains 1.7% of the catalytic activity for the synthesis of carbamoyl phosphate relative to the wild type CPS	Escherichia coli
A23K	A23K mutation decreases the glutamine-dependent ATPase activity by an order of magnitude. While there is a decrease in the rate of carbamoyl phosphate formation, the enzyme utilizes two molecules of ATP for every molecule of carbamoyl phosphate synthesized	Escherichia coli
G575F	mutant designed to block the migration of carbamate through the narrowest parts of the carbamate tunnel. Mutant retains 3.8% of the catalytic activity for the synthesis of carbamoyl phosphate relative to the wild type CPS	Escherichia coli
G575F	mutation to G575 does not exhibit significant pertubations to the kinetic constants of the partial reactions, G575F mutant has a 50fold reduction in the rate of carbamoyl phosphate formation (attributed to the restricted passage of carbamate through the tunnel). The insertion of a larger phenylalanine side chain is anticipated to create a more efficient blockage of the tunnel. Of the mutants made in the carbamate tunnel, G575F is the most efficacious at blocking the passage of carbamate without disrupting any of the active sites	Escherichia coli
G575K	mutation to G575 does not exhibit significant pertubations to the kinetic constants of the partial reactions, mutant has similar catalytic properties as the wild-type protein, suggesting that the conformational change of Arg-848 may not be crucial for the transport of carbamate	Escherichia coli
I18W/A23F/C24F	triple mutant I18W/A23F/C24F is made to disrupt the water pocket that may facilitate the passage of carbamate through the carbamate tunnel. This mutant significantly hinders the overall rate of carbamoyl phosphate synthesis and it diminishes all of the other partial reactions	Escherichia coli
L648E	mutant shows no detectable rate of carbamoyl phosphate formation. Little effect on the rates of all partial reactions is observed. Thus the reactions at the small subunit and the carboxy phosphate active sites remains unperturbed. The L648E mutant exhibits a 10fold drop in the rate of the glutaminase reaction which is due to the uncoupling between the carboxy phosphate and glutaminase active sites	Escherichia coli
L720E	the rates for both the glutamine- and HCO3--dependent ATPase reactions are largely unaffected by the mutation. The rate of the partial ATP-synthesis reaction is decreased 4fold. These perturbations may be due to an altered active site environment which diminishes the rate ADP phosphorylation by carbamoyl phosphate. No carbamoyl phosphate formation is detected. Mutant can structurally block the exit of the carbamate tunnel although the presence of the glutamate also weakens the assistance of Arg-848 during the synthesis of carbamoyl phosphate	Escherichia coli
M174E	mutant has significant reductions in the rates of the ATPase and carbamoyl phosphate synthesis reactions. Mutation does not affect the rate of the partial ATP synthesis reaction	Escherichia coli
M174E/M378E	catalytic properties of the double mutant, M174E/M378E, are similar to the single mutants with regard to the various partial reactions. Double mutant is unable to synthesize carbamoyl phosphate	Escherichia coli
M378E	mutant has significant reductions in the rates of the ATPase and carbamoyl phosphate synthesis reactions. Mutation does not affect the rate of the partial ATP synthesis reaction	Escherichia coli
M911E	the rate for both the glutamine- and HCO3--dependent ATPase reactions are largely unaffected by the mutation. The rate of the partial ATP-synthesis reaction is virtually undetectable. This perturbation may be due to an altered active site environment which diminishes the rate ADP phosphorylation by carbamoyl phosphate. Only limited carbamoyl phosphate formation is detected with mutant M911E. M911E mutant can structurally block the exit of the carbamate tunnel although the presence of glutamate also weakens the assistance of Arg-848 during the synthesis of carbamoyl phosphate	Escherichia coli

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.013	-	ATP	mutant G575K, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.014	-	ATP	mutant G575F, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.014	-	ATP	mutant G575F, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.022	-	ADP	mutant A23K, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.03	-	ADP	mutant G575F, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.03	-	ATP	mutant M378E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.051	-	ATP	wild-type, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.054	-	ADP	mutant G575K, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.07	-	ATP	mutant G575K, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.088	-	ADP	mutant M174E, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.1	-	L-Gln	mutant A23K, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.12	-	ATP	mutant A23F, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.13	-	ADP	mutant M378E, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.13	-	ADP	wild-type, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.15	-	L-Gln	wild-type, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.16	-	L-Gln	mutant I18W/A23F/C24F, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.16	-	ATP	mutant M378E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.17	-	L-Gln	mutant G575K, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.17	-	ADP	mutant M174E/M378E, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.18	-	ADP	mutant I18W/A23F/C24F, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.18	-	L-Gln	mutant L720E, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.18	-	L-Gln	mutant M911E, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.19	-	L-Gln	mutant G575F, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.21	-	L-Gln	mutant A23F, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.21	-	ATP	mutant I18W/A23F/C24F, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.22	-	ATP	mutant L720E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.23	-	L-Gln	mutant L648E, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.24	-	ATP	wild-type, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.25	-	ATP	mutant I18W/A23F/C24F, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.26	-	ATP	mutant M911E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.28	-	ATP	mutant M174E/M378E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.29	-	ATP	mutant M174E/M378E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.33	-	ATP	mutant A23K, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.34	-	ADP	mutant L648E, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.35	-	L-Gln	mutant M378E, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.36	-	ATP	mutant M174E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.38	-	ATP	mutant A23K, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.39	-	L-Gln	mutant M174E, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.42	-	ADP	mutant L720E, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.47	-	L-Gln	mutant M174E/M378E, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.5	-	ATP	mutant L648E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.53	-	ADP	mutant A23F, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.69	-	ATP	mutant M174E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
1	-	ATP	mutant M911E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
1.2	-	ATP	mutant L648E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
1.6	-	ATP	mutant A23F, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
1.7	-	ATP	mutant L720E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	-	-	-
Escherichia coli	P00968	large subunit	-

Reaction

Reaction	Comment	Organism	Reaction ID
2 ATP + L-glutamine + hydrogencarbonate + H2O = 2 ADP + phosphate + L-glutamate + carbamoyl phosphate	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. The tunnel is composed of three continuous water pockets and two narrow connecting parts, near residues A23 and G575. The two narrow parts render two free energy barriers of 6.7 and 8.4 kcal/mol, respectively. Three water pockets are 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 R306 to rotate toward E25, E383, and E604. 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	Escherichia coli	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
2 ATP + L-Gln + HCO3- + H+ + H2O	-	Escherichia coli	2 ADP + phosphate + L-glutamate + carbamoyl phosphate	-	?

Synonyms

Synonyms	Comment	Organism
Carbamoylphosphate synthetase	-	Escherichia coli
CPS	-	Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.006	-	ATP	mutant I18W/A23F/C24F, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.01	-	ATP	mutant I18W/A23F/C24F, value below 0.01, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication	Escherichia coli
0.01	-	ATP	mutant L648E,value below 0.01, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication	Escherichia coli
0.01	-	ATP	mutant L720E, value below 0.01, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication	Escherichia coli
0.01	-	ATP	mutant M174E/M378E, value below 0.01, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication	Escherichia coli
0.01	-	ADP	mutant M911E, value below 0.01, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.012	-	ATP	mutant M378E, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication	Escherichia coli
0.013	-	ATP	mutant M378E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.015	-	ATP	mutant M911E, value below 0.01, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication	Escherichia coli
0.024	-	ADP	mutant L648E, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.028	-	ATP	mutant M174E/M378E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.031	-	ADP	mutant L720E, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.033	-	ATP	mutant A23F, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication	Escherichia coli
0.035	-	ATP	mutant M378E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.039	-	ATP	mutant I18W/A23F/C24F, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.043	-	ATP	mutant M174E/M378E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.053	-	ATP	mutant M174E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.058	-	ADP	mutant G575F, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.072	-	ATP	mutant G575F, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication	Escherichia coli
0.086	-	ADP	mutant I18W/A23F/C24F, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.097	-	ATP	mutant A23F, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.1	-	ADP	mutant M174E, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.1	-	L-Gln	mutant M174E/M378E, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.11	-	ATP	mutant M174E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.13	-	L-Gln	mutant I18W/A23F/C24F, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.13	-	L-Gln	mutant M378E, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.13	-	ADP	wild-type, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.14	-	L-Gln	mutant M911E, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.15	-	ADP	mutant G575K, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.15	-	L-Gln	mutant M174E, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.16	-	ATP	mutant A23K, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication	Escherichia coli
0.17	-	ATP	mutant A23K, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.17	-	ATP	wild-type, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.18	-	ATP	mutant G575K, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.19	-	ATP	mutant G575F, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.2	-	ADP	mutant M378E, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.21	-	L-Gln	mutant A23K, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
0.22	-	ADP	mutant M174E/M378E, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.23	-	ADP	mutant A23F, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.28	-	ATP	mutant A23K, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.32	-	ADP	mutant A23K, ATP-synthesis, pH 7.6, temperature not specified in the publication	Escherichia coli
0.33	-	ATP	mutant M911E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.39	-	ATP	mutant L720E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.5	-	ATP	mutant M911E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.59	-	ATP	mutant L648E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
0.84	-	L-Gln	mutant L720E, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
1.08	-	L-Gln	mutant A23F, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
1.3	-	ATP	mutant A23F, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
1.3	-	L-Gln	mutant L648E, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
1.9	-	ATP	mutant G575K, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication	Escherichia coli
1.9	-	ATP	mutant M174E, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication	Escherichia coli
1.9	-	ATP	wild-type, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication	Escherichia coli
2	-	ATP	mutant L720E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
2.2	-	L-Gln	wild-type, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
3	-	L-Gln	mutant G575F, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
3.1	-	L-Gln	mutant G575K, glutaminase activity pH 7.6, temperature not specified in the publication	Escherichia coli
3.2	-	ATP	mutant L648E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
3.3	-	ATP	mutant G575F, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
3.3	-	ATP	mutant G575K, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli
3.6	-	ATP	wild-type, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication	Escherichia coli