A structural and functional evaluation of the allosteric response of aspartate transcarbamoylase from Erwinia herbicola

Abstract

aspartate transcarbamoylases (EC 2.1.3.2, ATCase) which are structurally similar, unique differences in homotropic and heterotropic regulation are present among the divergent species. The ATCase holoenzyme from Escheiichia coli is activated by ATP and inhibited by CTP; its CTP inhibition is synergistically enhanced in the presence of UTP. The ATCase holoenzyme from Erwinia herbicola is not affected by ATP and only slightly inhibited by CTP, yet it is synergistically inhibited by CTP in the presence of UTP. The ATCase holoenzyme from Proteus vulgafis is activated by ATP and CTP, yet it is synergistically inhibited by CTP in the presence of UTP. In this study, hybrid enzymes have been heterologously assembled in vivo using the catalytic and regulatory subunits from E. herbicola with catalytic and regulatory subunits from E. coli and Proteus vulgatis. The regulatory dimers tended to dictate the nature of the allosteric response in mixed hybrid enzymes. Holoenzymes formed with regulatory dimers from E. herbicola exhibited no response to ATP, inhibition by CTP, and synergistic inhibition by CTP in the presence of UTP. CTP inhibition was greater in the hybrid holoenzymes than that found for the native E. herbicola enzyme. The hybrid holoenzyme assembled in vivo with catalytic subunits from E. herbicola and regulatory subunits from E. coli exhibited activation by ATP, inhibition by CTP, and synergistic inhibition to CTP in the presence of UTP. These observations mimick the allosteric response of the native E. coli holoenzyme. The hybrid holoenzyme assembled in vivo with catalytic subunits from E. herbicola and regulatory subunits from P. vulgaris exhibited activation by ATP and CTP, and no effect by CTP in the presence of UTP. These heterologous effects are consistent with the allosteric response of the native P. vulgaiis holoenzyme. However, this hybrid holoenzyme lacked synergistic inhibition of CTP in the presence of UTP while UTP did counteract the activation of CTP.