LIMITED PROTEOLYSIS OF THE BETA-2-DIMER OF TRYPTOPHAN SYNTHASE YIELDS AN ENZYMATICALLY ACTIVE DERIVATIVE THAT BINDS ALPHA-SUBUNITS

Proteolytic modification of tryptophan synthase holo-beta-2-subunit from Escherichia coli at the C-terminal side of E-296 leads to an active species (E-296-nicked holo-beta-2) capable of interacting with alpha-subunits. Although this heterologous subunit interaction is rather weak, it induces an increase in catalytic efficiency in E-296-nicked holo-beta-2 by a factor of about 150. Correspondingly, enzymatic activity of alpha-subunits is enhanced 180-fold. This is in striking contrast to the findings from earlier reports which demonstrated that proteolytic derivatives modified at other positions in the "hinge region" embedded in the C-domain of the beta-2-subunit (K-272, R-275, and K-283) are enzymatically inactive and cannot associate with alpha-subunits. The equilibrium binding curve for the cofactor pyridoxal 5'-phosphate to E-296-nicked apo-beta-2 is hyperbolic (i.e., noncooperative), yielding an apparent microscopic dissociation constant, K(d), of 5 X 10(-6) M. This value closelyresembles the low-affinity dissociation constant of cooperative cofactor binding to the native beta-2-subunit, indicating that the conformational interactions between structural domains in the modified beta-protein seem to be disturbed considerably.