title: The tryptophan synthase alpha 2 beta 2 complex: kinetic studies with a mutant enzyme (beta K87T) to provide evidence for allosteric activation by an aminoacrylate intermediate.
abstract: To investigate the mechanism by which the tryptophan synthase beta subunit accelerates the cleavage of the indole-3-glycerol phosphate catalyzed by the alpha subunit (alpha reaction), kinetic experiments were carried out with wild-type and mutant forms of the alpha 2 beta 2 complex. Previous studies indicate that this activation can be attributed to the conformational changes associated with the formation of a Schiff base between aminoacrylate and pyridoxal phosphate at the beta site. To test this hypothesis, we investigated a mutant form of the alpha 2 beta 2 complex having the lysine-87 of its beta subunits replaced by threonine. The mutant alpha 2 beta 2 complex (K87T) exhibits normal activity for the alpha reaction but fails to catalyze formation of L-tryptophan from L-serine and indole (beta reaction). However, the mutant enzyme can form a Schiff base intermediate with L-serine at the beta site. Using a "chemical rescue" method, we converted K87T L-serine intermediate to an aminoacrylate intermediate. Steady-state kinetic studies reveal that the aminoacrylate derivative exhibits a 7-fold enhancement in kcat/Km for the alpha reaction relative to that of the L-serine derivative of the mutant or the wild-type enzyme in the absence of L-serine. Rapid kinetic data show that the aminoacrylate derivative of the mutant enzyme exhibits a 6-fold increase in the rate constant for the indole-3-glycerol phosphate cleavage reaction. In addition, rate constants for the reverse reaction and product release steps are also altered. Together, these changes lead to a decrease in Km and an increase in kcat.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: U
authors.initials: U
authors.lastname: Banik
authors.forename: D M
authors.initials: DM
authors.lastname: Zhu
authors.forename: P B
authors.initials: PB
authors.lastname: Chock
authors.forename: E W
authors.initials: EW
authors.lastname: Miles
mesh_terms: D000179:Acrylates / Q000378:metabolism*; D000494:Allosteric Regulation; D004789:Enzyme Activation; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D014367:Tryptophan Synthase / Q000235:genetics / Q000378:metabolism*
chemical_list: D000179:Acrylates; D014367:Tryptophan Synthase