Characterization of the pyrB gene of Serratia marcescens and hybrid gene formation with the pyrB gene of Escherichia coli, leading to the production of chimeric ATCases

Typescript (photocopy).The ATCase from Escherichia coli has been the subject of extensive physical, biochemical and genetic studies. The exact natures of the intrasubunit and intersubunit interactions responsible for enzymatic function are still not completely defined. While many residues have been implicated from chemical modification studies as participating in substrate binding, heterotropic interactions or homotropic interactions, x-ray crystallographic studies have suggested that some of these amino acids may not be directly involved. Site-directed mutagenesis of the genes that encode the subunits of ATCase can potentially define more directly residues that are involved in structural and functional roles of the enzyme, although it is hardly feasible to replace all the residues of ATCase with all other possible amino acid residues. In this study, the nucleotide and deduced amino acid sequences of the catalytic polypeptide of ATCase from the enteric bacterium Serratia marcescens HY will be investigated. The polypeptide contains 305 amino acids, as compared to 310 in the corresponding polypeptide from E. coli, and 40 amino acid differences from the E. coli polypeptide, counting gaps as mismatches. Hybrid pyrB genes have been constructed from portions of the pyrB genes of E. coli and S. marcescens. These hybrid pyrB genes produce hybrid catalytic polypeptides which have been utilized to produce chimeric holoenzymes. These constructs implicate the equatorial domain of the catalytic polypeptide of S. marcescens as responsible for some of the altered characteristics seen in these hybrid and chimeric ATCases. One chimeric holo-ATC case, C[subscript EC:Sm]R[subscript Ec], has an abnormally high [S][subscript 0.5] for aspartate of 125mM, although it retains characteristic E. coli-type ATP activation and CTP inhibition. The C[subscript Ec:Sm]R[subscript Sm] chimera exibits a hyperbolic dependence upon aspartate, with a K[subscript m] of 3.5 mM aspartate. The enzyme retains heterotopic responses, although the observed ATP and CTP imbibition is not characteristic of the enzyme from other species. The C[subscript Sm:Ec]R[subscript Ec] chimera exhibits a sigmoidal dependence on aspartate, with [S][subscript 0.5] of 11.5mM. The enzyme retains characteristic E. coli CTP inhibition and ATP activation. While most chimeric constructs exhibit the effector responses characteristic of the regulatory subunits present, the C[subscript Ec:Sm]R[subscript Sm] chimeric holoenzyme indicates that the allosteric response may be partially dependent upon interactions between the equatorial domain of the catalytic polypeptide and regulatory subunits. A comparison of these enzymatic and regulatory differences, coupled with the amino acid sequence differences may help to further identify components of ATCase that are necessary for its homotropic and heterotropic characteristics