THE TRYPTOPHAN SYNTHASE ALPHA-2-BETA-2 COMPLEX - KINETIC-STUDIES WITHA MUTANT ENZYME (BETA-K87T) TO PROVIDE EVIDENCE FOR ALLOSTERIC ACTIVATION BY AN AMINOACRYLATE INTERMEDIATE

TO investigate the mechanism by which the tryptophan synthase beta sub unit accelerates the cleavage of the indole-3-glycerol phosphate catal yzed by the alpha subunit (alpha reaction), kinetic experiments were c arried out with wild-type and mutant forms of the alpha(2) beta(2) com plex. Previous studies indicate that this activation can be attributed to the conformational changes associated with the formation of a Schi ff 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 norm al activity for the a reaction but fails to catalyze formation of L-tr yptophan 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 i ntermediate to an aminoacrylate intermediate. Steady-state kinetic stu dies reveal that the aminoacrylate derivative exhibits a 7-fold enhanc ement in k(cat)/K-m for the alpha reaction relative to that of the L-s erine 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, ra te constants for the reverse reaction and product release steps are al so altered. Together, these changes lead to a decrease in K-m and an i ncrease in k(cat). The magnitude of this enhancement is lower than tha t observed with the wild-type enzyme with saturating L-serine; neverth eless, the results directly demonstrate the postulated allosteric acti vation in the absence of L-tryptophan formation at the beta site.