LYSINE-87 IN THE BETA-SUBUNIT OF TRYPTOPHAN SYNTHASE THAT FORMS AN INTERNAL ALDIMINE WITH PYRIDOXAL-PHOSPHATE SERVES CRITICAL ROLES IN TRANSIMINATION, CATALYSIS, AND PRODUCT RELEASE

This study provides valuable insights into the functions of the lysine residue that forms an internal aldimine with pyridoxal phosphate in t he beta subunit of tryptophan synthase from Salmonella typhimurium. Ou r spectroscopic and kinetic studies demonstrate that a mutant alpha2be ta2 complex having beta subunit lysine 87 replaced by threonine forms external aldimines with several amino acids including L-serine, beta-c hloro-1-alanine, L-tryptophan, and D-tryptophan. Because the rates of aldimine formation are very slow, we conclude that one role of lysine 87 in the wild type enzyme is to facilitate formation of external aldi mines by transimination. Lysine 87 is an essential catalytic residue b ecause the mutant alpha2beta2 complex has no measurable activity in re actions catalyzed by the beta subunit and does not convert external al dimines to products. The mutant enzyme carries out two slow partial be ta-elimination reactions: the conversion of beta-chloro-L-alanine and L-serine to enzyme-bound aminoacrylate. The reaction with L-serine is catalyzed by ammonia, which partially replaces the deleted epsilon-ami no group. Lysine 87 is important for substrate and product release bec ause L-serine, L-tryptophan, and aminoacrylate dissociate very slowly from the mutant alpha2beta2 complex. Our ability to prepare very stabl e derivatives of the mutant alpha2beta2 complex containing tightly bou nd aldimines with a substrate, a product, or a reaction intermediate p rovides valuable materials for ongoing x-ray crystallographic investig ations and future kinetic analyses of the allosteric activation of the alpha subunit by beta subunit ligands.