Conversion of the allosteric regulatory patterns of aspartate transcarbamoylase by exchange of a single beta-strand between diverged regulatory chains.

Although structurally very similar, the aspartate transcarbamoylases (ATCase) of Serratia marcescens and Escherichia coli differ in both regulatory and catalytic characteristics. Most notably, CTP stimulates the catalytic activity of the S. marcescens ATCase and CTP/UTP inhibitory synergism has been lost. These allosteric characteristics contradict the traditional logic developed from the E. coli enzyme in which CTP and UTP function together as end products of the pyrimidine pathway to allosterically control the catalytic activity. In this study, five divergent residues (r93-r97) of the regulatory polypeptide of the S. marcescens enzyme have been replaced with their E. coli counterparts. These residues correspond to the S5' beta-strand of the allosteric effector binding domain at the junction of the allosteric and zinc domains of the regulatory polypeptide. In spite of the fact that the chimeric ATCase (SM:rS5'ec) retained 455 out of 460 amino acids of the S. marcescens enzyme, it possessed characteristics similar to those of the E. coli enzyme: (1) the [Asp]0.5 decreased from 40 to 5 mM; (2) ATP activation of the enzyme was greatly reduced; (3) CTP was converted from a strong activator to a strong inhibitor; and (4) the synergistic inhibition by CTP and UTP was restored. The S5' beta-strand is located at the outer surface of a five-stranded beta-sheet of the allosteric domain, providing a potential structural mechanism defining the allostery of this enzyme.