Mechanism of activation of the tryptophan synthase alpha2beta2 complex. Solvent effects of the co-substrate beta-mercaptoethanol.
To characterize the conformational transitions that lead to activation of catalysis by the tryptophan synthase alpha2beta2 complex, we have determined the solvent effects of a co-substrate, beta-mercaptoethanol, and of a model nonsubstrate, ethanol, on the catalytic and spectroscopic properties of the enzyme. Our results show that ethanol and beta-mercaptoethanol both alter the equilibrium distribution of pyridoxal 5'-phosphate intermediates formed in the reactions of L-serine at the beta site in the alpha2beta2 complex. Addition of increasing concentrations of ethanol increases the proportion of the external aldimine of L-serine and decreases the proportion of the external aldimine of aminoacrylate. Low concentrations of the co-substrate beta-mercaptoethanol (Kd = approximately 13 mM) decrease the proportion of the external aldimine of aminoacrylate and induce formation of the quinonoid of S-hydroxyethyl-L-cysteine. Higher concentrations of beta-mercaptoethanol decrease the concentration of the quinonoid intermediate and increase the proportion of the external aldimine of L-serine. Data analysis shows that beta-mercaptoethanol and ethanol both interact or bind preferentially 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 subunit, possibly the hydrophobic indole tunnel, becomes less exposed to solvent in the conformational transition that activates the alpha2beta2 complex.
Laboratory of Biochemical Pharmacology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
S A
SA
Ahmed
P
P
McPhie
E W
EW
Miles
D000494:Allosteric Regulation; D004789:Enzyme Activation; D000431:Ethanol / Q000737:chemistry; D008623:Mercaptoethanol / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity; D014367:Tryptophan Synthase / Q000378:metabolism*
D012997:Solvents; D000431:Ethanol; D012694:Serine; D008623:Mercaptoethanol; D014367:Tryptophan Synthase