MECHANISM OF ACTIVATION OF THE TRYPTOPHAN SYNTHASE ALPHA(2)BETA(2) COMPLEX - SOLVENT EFFECTS OF THE COSUBSTRATE BETA-MERCAPTOETHANOL

To characterize the conformational transitions that lead to activation of catalysis by the tryptophan synthase alpha(2) beta(2), complex, we have determined the solvent effects of a co-substrate, beta-mercaptoe thanol, and of a model nonsubstrate, ethanol, on the catalytic and spe ctroscopic properties of the enzyme. Our results show that ethanol and beta-mercaptoethanol both alter the equilibrium distribution of pyrid oxal 5'-phosphate intermediates formed in the reactions of L-serine at the beta site in the alpha(2) beta(2) complex. Addition of increasing concentrations of ethanol increases the proportion of the external al dimine of L-serine and decreases the proportion of the external aldimi ne of aminoacrylate. Low concentrations of the co-substrate beta-merca ptoethanol (K-d = similar to 13 mM) decrease the proportion of the ext ernal aldimine of aminoacrylate and induce formation of the quinonoid of S-hydroxyethyl-L-cysteine. Higher concentrations of beta-mercaptoet hanol decrease the concentration of the quinonoid intermediate and inc rease the proportion of the external aldimine of L-serine. Data analys is shows that beta-mercaptoethanol and ethanol both interact or bind p referentially with the conformer of the enzyme that predominates when the aldimine of L-serine is formed and shift the equilibrium in favor of this conformer. We propose that a nonpolar region of the beta subun it, possibly the hydrophobic indole tunnel, becomes less exposed to so lvent in the conformational transition that activates the alpha(2) bet a(2) complex.