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, Kd, of 5 x 10(-6) M. This value closely resembles 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.
Kaufmann
M
M
Institut für Physiologische Chemie, Universität Witten/Herdecke, Germany.
Schwarz
T
T
Jaenicke
R
R
Schnackerz
K D
KD
Meyer
H E
HE
Bartholmes
P
P
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology; D014367:Tryptophan Synthase / Q000378:metabolism*
D011732:Pyridoxal Phosphate; D014367:Tryptophan Synthase