A structural and functional evaluation of the allosteric response of the Aspartate transcarbamoylase from Serratia marcescens

Abstract

The aspartate transcarbamoylases (E.C.2.1.3.2, ATCase) from enteric bacteria possess structural and functional similarities, although unique differences in homotropic and heterotropic regulation of enzymatic activity exist among divergent species. The ATCase holoenzyme from Escherichia coli is activated by ATP, inhibited by CTP, and synergistically inhibited by CTP in the presence of UTP. The ATCase holoenzyme from Serratia marcescens is activated by ATP and by CTP, while a combination of CTP and UTP counteracts the activation of CTP alone. The genes encoding the catalytic and regulatory polypeptides of the ATCase from S. marcescens, pyrB and pyrI, are arranged as a bicistronic operon with pyrI promoter distal. A comparison of the DNA sequences of pyrI from E. coli and S. marcescens reveals a 75% identity, and the deduced amino acid homology is 77%. The regulatory polypeptide from S. marcescens contains 154 amino acids with a molecular mass of 17,308 Da. In vivo hybrid enzymes have been assembled with catalytic subunits from divergent enteric species and regulatory subunits from S. marcescens. The regulatory dimers of S. marcescens dictate the nature of the allosteric response to the catalytic trimers. In each of the enzymes, the UTP response is either negligible or significantly less than the activation by both ATP and CTP; moreover, a combination of CTP and UTP counteracts the activation of CTP. These observations are consistent with the allosteric response of the native S. marcescens holoenzyme. Chimeric regulatory polypeptides can be made by genetically exchanging the allosteric domain and the zinc domain from E. coli and S. marcescens in both combinations. Using catalytic polypeptides from both species and both chimeric regulatory polypeptides, a series of active chimeric holoenzymes have been assembled in vivo. ATP uniformly activates the chimeric enzymes, but the CTP response is variable, indicating that ATP and CTP exert heterotropic effects through a different series of amino acid contacts. Residues within the allosteric domain of the S. marcescens regulatory polypeptide cannot transmit CTP effects through the E. coli zinc domain to be interpreted by the holoenzyme; however, the zinc domain of S. marcescens interprets CTP effects from either the E. coli allosteric domain or the S. marcescens allosteric domain.