Literature summary for 2.1.3.2 extracted from

Cockrell, G.M.; Kantrowitz, E.R.
Metal ion involvement in the allosteric mechanism of Escherichia coli aspartate transcarbamoylase (2012), Biochemistry, 51, 7128-7137.

Cloned(Commentary)

Cloned (Comment)	Organism
overexpression in Escherichia coli strain EK1104 transformed with plasmid pEK15212 containing the Escherichia coli pyrBI gene	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant ATCase in complex with UTP, CTP, or dCTP, dialysis of 20 mg/ml protein against 40 mM sodium citrate, 1 mM 2-mercaptoethanol, 0.2 mM EDTA, and 1.0 mM CTP, pH 5.7, at 20°C, 1 week, transfer of dialysis buttons to 2 mL of crystallization buffer with 5 mM UTP and 5 mM MgCl2 and equilibration for 12 h, and to crystallization buffer containing 5 mM UTP and 5 mM MgCl2 for 12 h, respectively, cryoprotection in 20% 2-methyl-2,4-pentanediol in UTP-Mg2+ crystallization buffer, X-ray diffraction structure determination and analysis at 1.9-2.1 A resolution	Escherichia coli

Crystallization (Comment)

Organism

purified recombinant ATCase in complex with UTP, CTP, or dCTP, dialysis of 20 mg/ml protein against 40 mM sodium citrate, 1 mM 2-mercaptoethanol, 0.2 mM EDTA, and 1.0 mM CTP, pH 5.7, at 20°C, 1 week, transfer of dialysis buttons to 2 mL of crystallization buffer with 5 mM UTP and 5 mM MgCl2 and equilibration for 12 h, and to crystallization buffer containing 5 mM UTP and 5 mM MgCl2 for 12 h, respectively, cryoprotection in 20% 2-methyl-2,4-pentanediol in UTP-Mg2+ crystallization buffer, X-ray diffraction structure determination and analysis at 1.9-2.1 A resolution

Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
D19A	a regulatory mutant, which does not exhibit UTP synergistic inhibition	Escherichia coli
H20A	a regulatory mutant, which does not exhibit UTP synergistic inhibition	Escherichia coli
K56A	a regulatory mutant, which does not exhibit UTP synergistic inhibition	Escherichia coli
K60A	a regulatory mutant, which does not exhibit UTP synergistic inhibition	Escherichia coli
K6A	a regulatory mutant, which does not exhibit UTP synergistic inhibition	Escherichia coli
L7A	a regulatory mutant, which does not exhibit UTP synergistic inhibition	Escherichia coli

Inhibitors

Inhibitors	Comment	Organism
CTP	demetaled CTP, synergistic inhibition with UTP, while UTP alone has little or no influence on the enzyme activity, mechanism, overview. Binding of UTP can enhance the binding of CTP and presence of a metal ion such as Mg2+ is required for synergistic inhibition. Structure of the ATCase-CTP-UTP-Mg2+ complex	Escherichia coli
dCTP	-	Escherichia coli
additional information	conformational changes due to nucleotide binding, overview	Escherichia coli
UTP	inhibition with CTP, while UTP alone has little or no influence on the enzyme activity, mechanism, overview. UTP, in the presence of dCTP or CTP, binds at a site on a regulatory side chain that does not overlap the CTP/dCTP site, and the triphosphates of the two nucleotides are parallel to each other with a metal ion, in this case Mg2+, coordinated between the beta- and gamma-phosphates of the two nucleotides. UTP binds more tightly in the presence of CTP. Structure of the ATCase-CTP-UTP-Mg2+ complex	Escherichia coli

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	allosteric mechanism with metal ion involvement, overview	Escherichia coli

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required for synergistic inhibition of the enzyme by CTP and UTP, metal binding site in the allosteric regulatory site of ATCase, overview	Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
L-aspartate + carbamoyl phosphate	Escherichia coli	-	phosphate + N-carbamoyl-L-aspartate	-	?

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P0A786	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant enzyme from Escherichia coli strain EK114 by anion exchange chromatography, ammonium sulfate fractionation, and hydrophobic interaction chromatography	Escherichia coli

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
L-aspartate + carbamoyl phosphate	-	Escherichia coli	phosphate + N-carbamoyl-L-aspartate	-	?
additional information	conformational changes due to nucleotide binding, overview	Escherichia coli	?	-	?

Synonyms

Synonyms	Comment	Organism
aspartate transcarbamoylase	-	Escherichia coli
ATCase	-	Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Escherichia coli

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Escherichia coli

General Information

General Information	Comment	Organism
metabolism	aspartate transcarbamoylase allosterically regulates pyrimidine nucleotide biosynthesis	Escherichia coli
physiological function	aspartate transcarbamoylase allosterically regulates pyrimidine nucleotide biosynthesis	Escherichia coli