title: State of Tyr49 in a mutant tryptophan synthase alpha-subunit substituted at position 49.
abstract: The states of tyrosine residues in an alpha-subunit of wild-type tryptophan synthase from Escherichia coli and a mutant protein which has tyrosine in place of glutamic acid at position 49, were examined by absorption spectrum, spectrophotometric titration of phenolic hydroxyl ionization, and deuteration kinetics of phenolic hydrogen monitored by fluorescence measurement. The difference absorption spectrum of the mutant protein against the wild-type protein at pH 7.0 and 25 degrees C had peaks at 289, 282, and 276.5 nm. These positions corresponded to those in the absorption spectrum of L-tyrosine derivatives in a non-aqueous solvent at 77 K and these bands were well-resolved even at 25 degrees C as if the tyrosine residue were fixed at lower temperature. The titration curve of the mutant protein at 3 degrees C differed from that of the wild-type protein only above pH 12.7, where the difference molar extinction coefficient at 295 nm reached a plateau, indicating that ionization of Tyr49 took place at an abnormally high pH. These results suggest that Tyr49 is buried in the hydrophobic interior and fixed in a certain orientation. The deuterium exchanges of phenolic hydrogen at pH 7.0 and 13 degrees C in the wild-type and mutant proteins consisted of a single and two first order processes, respectively, all three having smaller rate constants than that of free tyrosine, indicating that these tyrosine residues are buried. It is concluded that Tyr49 in the mutant protein is not on the surface of the molecule.
mesh_terms: D055598:Chemical Phenomena; D002621:Chemistry; D002942:Circular Dichroism; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D014367:Tryptophan Synthase / Q000235:genetics*; D014443:Tyrosine
chemical_list: D014443:Tyrosine; D003903:Deuterium; D014367:Tryptophan Synthase