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A more recent version of this article appeared on June 28, 2002
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Papers In Press, published online ahead of print April 16, 2002
J. Biol. Chem, 10.1074/jbc.M200009200
Submitted on January 2, 2002
Revised on March 13, 2002
Accepted on April 16, 2002

Pseudomonas aeruginosa aspartate transcarbamoylase: characterization of its catalytic and regulatory properties

John F. Vickrey, Guy Hervé, and David R. Evans

Department of Biochemistry and Molecular Biology, Wayne State University, Detroit, MI 48201

Corresponding Author: devans{at}cmb.biosci.wayne.edu

Aspartate transcarbamoylase from Pseudomonadaceae is 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. ATCase from Pseudomonas aeruginosa was cloned and over-expressed in E. coli. The homogenous His-tagged protein could be isolated in high yield, 30 mg/liter of culture, by affinity chromatography and crystallized. Attempts to dissociate the catalytic and pDHO subunits or to express catalytic subunits in the absence of pDHO 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 pyrimidine and purine 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 the 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 on the enzyme 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.


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