Properties of hybrid aspartate transcarbamoylase formed with native subunits from divergent bacteria

The aspartate transcarbamoylases (ATCase) from Serratia marcescens and Escherichia coli exhibit unique regulatory and kinetic properties and were dissociated into their constituent regulatory and catalytic subunits. A hybrid ATCase holoenzyme was formed with catalytic subunits from S. marcescens and regulatory subunits from E. coli as demonstrated by the molecular weight, the recovery of cooperative, homotropic response to the substrate aspartate, and the re-establishment of heterotropic responses to the allosteric effectors ATP and CTP. This hybrid is of interest since ATCase from E. coli is inhibited by CTP and activated by ATP while ATCase from S. marcescens is activated by both nucleotides. The activity of the catalytic subunits was reduced upon formation of the catalytic subunits was reduced upon formation of the hybrid ATCase enzyme which exhibited an exaggerated requirement for aspartate; the [S]0.5 was 100-125 mM aspartate compared to 5 mM for the E. coli holoenzyme and 20 mM for the native ATCase from S. marcescens. Still, the heterotropic response to effectors was communicated efficiently through the various protein:protein domains of bonding in ATCase as 1 mM ATP activated the hybrid ATCase while 1 mM CTP inhibited the enzyme. ATP did not influence the pH profile of the hybrid enzyme but increasing concentrations of the substrate aspartate shifted the pH optimum from pH 6 to pH 6.8. These data support the view that homotropic and heterotropic responses in ATCase can be altered separately. Since the hybrid ATCase was formed with native, unmodified regulatory and catalytic subunits, the r:r and c:c domains in the hybrid holoenzyme remained fundamentally unaltered. Therefore, it appears that the r:c domains provide the primary communication for changes in quaternary structure that define the allosteric and enzymatic properties of the holoenzyme.