Three of the six possible intersubunit stabilizing interactions involving Glu-239 are sufficient for restoration of the homotropic and heterotropic properties of Escherichia coli aspartate transcarbamoylase.

A hybrid version of Escherichia coli aspartate transcarbamoylase was investigated in which one catalytic subunit has the wild-type sequence, and the other catalytic subunit has Glu-239 replaced by Gln. Since Glu-239 is involved in intersubunit interactions, this hybrid could be used to evaluate the extent to which T state stabilization is required for homotropic cooperativity and for heterotropic effects. Reconstitution of the hybrid holoenzyme (two different catalytic subunits with three wild-type regulatory subunits) was followed by separation of the mixture by anion-exchange chromatography. To make possible the resolution of the three holoenzyme species formed by the reconstitution, the charge of one of the catalytic subunits was altered by the addition of six aspartic acid residues to the C terminus of each of the catalytic chains (AT-C catalytic subunit). Control experiments indicated that the AT-C catalytic subunit as well as the holoenzyme formed with AT-C and wild-type regulatory subunits had essentially the same homotropic and heterotropic properties as the native catalytic subunit and holoenzyme, indicating that the addition of the aspartate tail did not influence the function of either enzyme. The control reconstituted holoenzyme, in which both catalytic subunits have Glu-239 replaced by Gln, exhibited no cooperativity, an enhanced affinity for aspartate, and essentially no heterotropic response identical to the enzyme isolated without reconstitution. The hybrid containing one normal and one mutant catalytic subunit exhibited homotropic cooperativity with a Hill coefficient of 1.4 and responded to the nucleotide effectors at about 50% of the level of the wild-type enzyme. Small angle x-ray scattering experiments with the hybrid enzyme indicated that in the absence of ligands it was structurally similar, but not identical, to the T state of the wild-type enzyme. In contrast to the wild-type enzyme, addition of carbamoyl phosphate induced a significant alteration in the scattering pattern, whereas the bisubstrate analog N-phosphonoacetyl-L-aspartate induced a significant change in the scattering pattern indicating the transition to the R-structural state. These data indicate that in the hybrid enzyme only three of the usual six interchain interactions involving Glu-239 are sufficient to stabilize the enzyme in a low affinity, low activity state and allow an allosteric transition to occur.