Amino acid substitutions which stabilize aspartate transcarbamoylase in the R state disrupt both homotropic and heterotropic effects.

We have used site-specific amino acid substitutions to investigate the linkage between the allosteric properties of arpartate transcarbamoylase and the global conformational transition exhibited by the enzyme upon binding active-site ligands. Two mutationally altered enzymes in which an amino acid substitution had been introduced at a single position in the catalytic polypeptide chain (Lys-164----Glu and Glu-239----Lys) and a third species harboring both of these substitutions (Lys-164:Glu-239----Glu:Lys) were constructed. Sedimentation velocity difference studies were performed in order to assess the effects of the amino acid substitutions on the quaternary structure of the holoenzyme in the absence and presence of various active-site ligands, including the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA), which has been shown previously to promote the allosteric transition. In the absence of ligand, two of the mutationally altered enzymes, Lys-164----Glu and Lys-164:Glu-239----Glu:Lys, existed in the R conformation, isomorphous with that of the PALA-liganded wild-type holoenzyme. These enzymes exhibited no conformational change upon binding PALA. The unliganded Glu-239----Lys enzyme had an average sedimentation coefficient intermediate between that of the unliganded and PALA-liganded states of the wild-type enzyme which could be accounted for in terms of a mixture of T- and R-state molecules. This mutant enzyme was converted to the fully swollen conformation upon binding PALA, phosphate or carbamoyl phosphate. The allosteric properties of the mutationally altered species were investigated by PALA-binding studies and by steady-state enzyme kinetics. In each case, the mutationally altered enzymes were devoid of both homotropic and heterotropic effects, supporting the premise that the allosteric properties of the wild-type enzyme are linked to a ligand-promoted change in quaternary structure.