Structural and functional interdependence of the protomers of Escherichia coli K 12 tryptophanase during binding of pyridoxal 5'-phosphate.

It has been shown previously that the binding of pyridoxal 5'-phosphate to Escherichia coli K 12 tryptophanase brings about an important conformational change of the protein. The way in which this structural change is transmitted from holoprotomers to apoprotomers is investigated here, using hybrid molecules (between apoprotomers and irreversibly saturated holoprotomers). It is shown that the binding of two or three coenzyme molecules per tetramer stabilizes the whole molecule against thermal inactivation and cold-induced dissociation. The change in conformation induced on an apoprotomer by the proximity of three holoprotomers is described, using three structural probes: the kinetics of binding of the cofactor and of 5'-phosphopyridoxyl-tryptophan (an analog of an intermediate in the catalytic reaction), and the reactivity of the essential cysteines. The kinetic anticooperatively in the binding of pyridoxal 5'-phosphate is confirmed, some of its parameters are determined, and its mechanism is interpreted in relation to the coupling between the protomers.