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.
Ahmed
S A
SA
Laboratory of Biochemical Pharmacology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
McPhie
P
P
Miles
E W
EW
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