Conformational changes and subunit communication in tryptophan synthase: effect of substrates and substrate analogs.
The transmission of regulatory signals between the alpha- and beta-subunits of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium has been investigated by monitoring the luminescence properties of the enzyme in the presence and in the absence of the alpha-subunit ligand DL-alpha-glycerol 3-phosphate, the alpha- and beta-subunit substrate indole, and the beta-subunit substrate analog L-histidine. The beta-subunit contains as intrinsic probes Trp-177 and pyridoxal 5'-phosphate, whereas the alpha-subunit has been mutagenized by replacing Ala-129 with a Trp residue. In contrast to the inertness of L-histidine, DL-alpha-glycerol 3-phosphate was found (i) to alter the phosphorescence spectrum of Trp-129, (ii) to shift the fluorescence thermal quenching profile of both Trp-177 and coenzyme to higher temperature, (iii) to slow down the triplet decay kinetics of Trp-177 in fluid solution, and (iv) to affect the equilibrium between different conformations of the enzyme. These findings provide direct evidence that DL-alpha-glycerol 3-phosphate binding affects the structure of the alpha-subunit and, in the presence of coenzyme, induces a conformational change in the beta-subunit that leads to a considerably more rigid structure. As opposed to DL-alpha-glycerol 3-phosphate, the shortening of the phosphorescence lifetime upon indole binding suggests that this substrate increases structural fluctuations in the beta-subunit. Implications for the mechanism of the allosteric regulation between alpha- and beta-subunits are discussed.
Strambini
G B
GB
CNR-Istituto di Biofisica, Pisa, Italy.
Cioni
P
P
Peracchi
A
A
Mozzarelli
A
A
D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D005798:Genes, Bacterial; D005994:Glycerophosphates / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D008163:Luminescent Measurements; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D013816:Thermodynamics; D014364:Tryptophan; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000378:metabolism*
D001051:Apoenzymes; D005994:Glycerophosphates; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006639:Histidine; D014364:Tryptophan; C029620:alpha-glycerophosphoric acid; D014367:Tryptophan Synthase