Pseudomonas aeruginosa aspartate transcarbamoylase. Characterization of its catalytic and regulatory properties.
Aspartate transcarbamoylase from Pseudomonadaceae is a class A enzyme consisting of six copies of a 36-kDa catalytic chain and six copies of a 45-kDa polypeptide of unknown function. The 45-kDa polypeptide is homologous to dihydroorotase but lacks catalytic activity. Pseudomonas aeruginosa aspartate transcarbamoylase was overexpressed in Escherichia coli. The homogeneous His-tagged protein isolated in high yield, 30 mg/liter of culture, by affinity chromatography and crystallized. Attempts to dissociate the catalytic and pseudo-dihydroorotase (pDHO) subunits or to express catalytic subunits only were unsuccessful suggesting that the pDHO subunits are required for the proper folding and assembly of the complex. As reported previously, the enzyme was inhibited by micromolar concentrations of all nucleotide triphosphates. In the absence of effectors, the aspartate saturation curves were hyperbolic but became strongly sigmoidal in the presence of low concentrations of nucleotide triphosphates. The inhibition was unusual in that only free ATP, not MgATP, inhibits the enzyme. Moreover, kinetic and binding studies with a fluorescent ATP analog suggested that ATP induces a conformational change that interferes with the binding of carbamoyl phosphate but has little effect once carbamoyl phosphate is bound. The peculiar allosteric properties suggest that the enzyme may be a potential target for novel chemotherapeutic agents designed to combat Pseudomonas infection.
Vickrey
John F
JF
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michiagan 48201, USA.
Herve
Guy
G
Evans
David R
DR
D001221:Aspartate Carbamoyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002455:Cell Division; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008970:Molecular Weight; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012265:Ribonucleotides / Q000494:pharmacology; D013379:Substrate Specificity
D011994:Recombinant Proteins; D012265:Ribonucleotides; D001221:Aspartate Carbamoyltransferase