C. Craig Hyde

Crystallographic and kinetic studies of the tryptophan synthase α2β2 complex with a mutation in β subunit lysine-87 that binds pyridoxal phosphate

To probe the mechanism of reactions catalyzed by the tryptophan synthase α2β2 complex, we have substituted threonine for β subunit lysine-87 that forms an internal aldimine with pyridoxal phosphate. Our finding that the mutant α2β2 complex (K87T) is inactive, but forms external aldimine complexes of pyridoxal phosphate with L-serine and L-tryptophan, provides evidence that lysine-87 serves critical roles in transimination, catalysis and product release. Kinetic studies demonstrate that formation of a stable aminoacrylate intermediate at the β site of the K87T α2β2 complex results in activation of the a subunit. Crystallographic analyses at -2 A resolution of the L-serine and L-tryptophan intermediates with the K87T α2β2 complex localize the substrate and product binding sites of the β subunit.