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.
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)
Banik
U
U
Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Zhu
D M
DM
Chock
P B
PB
Miles
E W
EW
D000179:Acrylates / Q000378:metabolism*; D000494:Allosteric Regulation; D004789:Enzyme Activation; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D014367:Tryptophan Synthase / Q000235:genetics / Q000378:metabolism*
D000179:Acrylates; D014367:Tryptophan Synthase