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

Summary

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 Å 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.