Ligand-mediated changes in the tryptophan synthase indole tunnel probed by nile red fluorescence with wild type, mutant, and chemically modified enzymes.
The bacterial tryptophan synthase alpha 2 beta 2 complex contains an unusual structural feature: an intramolecular tunnel that channels indole from the active site of the alpha subunit to the active site of the beta subunit 25 A away. Here we investigate the role of the tunnel in communication between the alpha and beta subunits using the polarity-sensitive fluorescent probe, Nile Red. Interaction of Nile Red in the nonpolar tunnel near beta subunit residues Cys-170 and Phe-280 is supported by studies with enzymes altered at these positions. Restricting the tunnel by enlarging Cys-170 by chemical modification or mutagenesis decreases the fluorescence of Nile Red by 30-70%. Removal of a partial restriction in the tunnel by replacing Phe-280 by Cys or Ser increases the fluorescence of Nile Red more than 2-fold. A binding site for Nile Red in this region near the pyridoxal phosphate coenzyme of the beta subunit is further supported by iodide quenching and fluorescence energy transfer experiments and by molecular modeling based on the three-dimensional structure of the alpha 2 beta 2 complex. Finally, studies using Nile Red as a sensitive probe of conformational changes in the tunnel reveal that allosteric ligands (alpha subunit) or active site ligands (beta subunit) decrease the fluorescence of Nile Red. We speculate that allosteric and active site ligands induce a tunnel restriction near Phe-280 that serves as a gate to control passage of indole through the tunnel.
Ruvinov
S B
SB
Laboratory of Biochemical Pharmacology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
Yang
X J
XJ
Parris
K D
KD
Banik
U
U
Ahmed
S A
SA
Miles
E W
EW
Sackett
D L
DL
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003062:Codon; D003196:Computer Graphics; D003198:Computer Simulation; D003545:Cysteine; D004926:Escherichia coli; D005456:Fluorescent Dyes; D007211:Indoles; D007454:Iodides; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010078:Oxazines; D010649:Phenylalanine; D017354:Point Mutation; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology*; D012694:Serine; D013050:Spectrometry, Fluorescence; D014364:Tryptophan; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000378:metabolism*
D003062:Codon; D005456:Fluorescent Dyes; D007211:Indoles; D007454:Iodides; D008024:Ligands; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D010078:Oxazines; D011994:Recombinant Proteins; D012694:Serine; D010649:Phenylalanine; D011732:Pyridoxal Phosphate; D014364:Tryptophan; D014367:Tryptophan Synthase; D003545:Cysteine; C044808:nile red