6.3.4.2: CTP synthase (glutamine hydrolysing)

A20R

mutation disrupts cytoophidium assembly

DELTA20

deletion of the N-terminal 20 amino acids alone is sufficient to disrupt cytoophidium assembly, mutant protein forms distinct cytoplasmic structures which appear as large clusters

K2E

mutation disrupts cytoophidium assembly

Y3E

mutation disrupts cytoophidium assembly

C379A

mutant enzyme is fully active with ammonia but has no glutamine-dependent activity, no inhibition by glutamate gamma-semialdehyde

648963

D107A

enzyme exhibits wild-type NH3-dependent activity and affinity for glutamine, but impaired glutamine-dependent CTP formation, affinity of the mutant enzyme for GTP is reduced 2-4fold

E103A

mutant enzyme exhibits no glutamine-dependent activity and is only partially active with NH3

G110A

affinity of the mutant enzyme for GTP is reduced 2-4fold, enzyme exhibits wild-type NH3-dependent activity and affinity for glutamine, but impaired glutamine-dependent CTP formation

G142A

P0A7E5

site-directed mutagenesis, inactive mutant with both ammonia and glutamine

690817

G143A

P0A7E5

site-directed mutagenesis, kcat/Km for ammonia-dependent and glutamine-dependent CTP formation by mutant G143A are reduced by 22fold and 16fold, respectively, compared to the wild-type enzyme. The mutant is able to form active tetramers in the presence of ATP and UTP

690817

G146A

P0A7E5

site-directed mutagenesis, kcat/Km for ammonia-dependent and glutamine-dependent CTP formation by mutant G143A are reduced by 1.4fold and 1.8fold, respectively, compared to the wild-type enzyme. The mutant is able to form active tetramers in the presence of ATP and UTP

690817

G351A

mutation increases lability of the enzyme, mutant enzyme is not overproduced because of apparent instability and proteolytic degradation

648980

G352C

mutation increases lability of the enzyme, mutation abolishes the capacity to form the covalent glutaminyl-cysteine379 catalytic intermediate, thus preventing glutamine amide transfer function, mutant enzyme is not overproduced because of apparent instability and proteolytic degradation

G352P

H118A

mutant enzyme exhibits no glutamine-dependent activity and is only partially active with NH3

K102A

mutant enzyme exhibits wild-type activity with respect to NH3 and glutamine

K297A

A0A140N932

replacement of lysine 297 by alanine does not affect NH3-dependent CTP formation, relative to wild-type CTPS, but reduces kcat for the glutaminase activity 78fold

672395

K306A

A0A140N932

replacement of lysine 306 by alanine reduces the rate of 2',3'-dialdehyde adenosine 5'-triphosphate-dependent inactivation (Kinact = 0.0058/sec, Ki = 3.7 mM) and reduces the apparent affinity for CTPS for both ATP and UTP by 2fold. The efficiency of K306A-catalyzed glutamine-dependent CTP formation is also reduced 2fold while near wild type activity is observed when NH3 is the substrate. These findings suggest that Lys 206 is not essential for ATP binding, but does play a role in bringing about the conformational changes that mediate interactions between ATP and UTP sites, and between the ATP-binding site and the glutamine amide transfer domain

672395

L109A

R104A

mutant enzyme exhibits no glutamine-dependent activity and is only partially active with NH3

R105A

enzyme exhibits wild-type NH3-dependent activity and affinity for glutamine, but impaired glutamine-dependent CTP formation

V349S

mutation increases lability of the enzyme

648980

E579A

HEK 293 cells: no effect on phosphorylation of CTPS1 by glycogen synthase kinase 3

S462A/T455A

S462A and T455A mutations result in a decreased CTP synthetase 1 phosphorylation which appear to be much less than of the individual mutant enzymes S462A or T455A

674816

S568A

2fold increase in Km value for UTP. Mutation of S568A significantly increases CTPS2 activity. The S568A mutation has a greater effect on the glutamine than ammonia-dependent activity

715549

S571A

4fold increase in Km value for UTP

715549

S571I

Ser-571 is the major site phosphorylated by glycogen synthase kinase 3 in intact human embryonic kidney 293 cells

S571I/S574A

phosphorylation by glycogen synthase kinase 3 does not show altered incorporation of phosphate compared with S571I alone

S571I/S574A/S575A

phosphorylation by glycogen synthase kinase 3 shows a slight decrease in the amount of phosphate incorporated into CTPS1 compared with S571I/S575A, suggesting that Ser-574 may serve as a minor secondary site for glycogen synthase kinase 3 phosphorylation

S571I/S575A

phosphorylation by glycogen synthase kinase 3 shows slightly elevated amounts of phosphate incorporated into CTPS1 compared with S571I, suggesting that without the ability to phosphorylate Ser-571 or Ser-575 in vitro, glycogen synthase kinase 3 may phosphorylate an alternative site, albeit to a much lesser extent

S574A

greatly reduces phosphorylation by glycogen synthase kinase 3

S574A/S575A

greatly reduces phosphorylation by glycogen synthase kinase 3

S575A

T455A

T455A mutation causes a 78% decrease in protein kinase A phosphorylation

674886

E362Q

turnover number for NH4Cl-dependent GTP synthesis reaction is 1.6fold higher than wild-type value

G360A

5fold increase in GTP-dependent activation of uncoupled glutamine hydrolysis compared to wild-type enzyme. Turnover number for NH4Cl-dependent GTP synthesis reaction is 1.2fold higher than wild-type value

G360P

mutant enzyme shows no GTP activation of the uncoupled glutaminase reaction, about 4fold lower turnover number for NH4Cl-dependent CTP synthesis reaction than wild-type enzyme

R359M

mutant enzyme shows no GTP activation of the uncoupled glutaminase reaction. Turnover number for NH4Cl-dependent GTP synthesis reaction is 1.1fold higher than wild-type value

R359P

mutant enzyme shows no GTP activation of the uncoupled glutaminase reaction. Turnover number for NH4Cl-dependent GTP synthesis reaction is 1.2fold lower than wild-type value

C404G

inactive

D70A

mutation in the ATP binding site, mutant exhibits approximately twofold increase in the number of cells containing filaments

E146A

mutation in the ATP binding site, mutant exhibits approximately twofold increase in the number of cells containing filaments

E161K

R381M

mutation in L11 lid, 3.1fold increase in the number of cells forming filaments

R381P

mutation in L11 lid, 3.1fold increase in the number of cells forming filaments

S330A

CTP synthetase activity in cells bearing the mutant enzyme is elevated, mutation causes an elevation in the Vmax of the reaction. Mutation does not have a major effect on the oligomerization of CTP synthetase

648973

S354A

CTP synthetase activity in extracts from cells bearing the mutant enzyme is reduced when compared with cells bearing the wild-type enzyme, decrease in Vmax of the reaction. The amount of inactive dimeric enzyme form is 98% greater compared to wild-type enzyme

648973

S36A

S454A

CTP synthetase activity in extracts from cells bearing the mutant enzyme is reduced when compared with cells bearing the wild-type enzyme. Mutation does not have a major effect on the oligomerization of CTP synthetase

648973

C404G

inactive

D70A

mutation in the ATP binding site, mutant exhibits approximately twofold increase in the number of cells containing filaments

E146A

mutation in the ATP binding site, mutant exhibits approximately twofold increase in the number of cells containing filaments

E161K

mutation in CTP binding site, filament formation is completely disrupted and the enzyme can only form foci. Mutation decreases the affinity of the enzyme for CTP

S36A