Cooperative binding of L-trp to human tryptophan 2,3-dioxygenase: resonance Raman spectroscopic analysis.
Tryptophan 2,3-dioxygenase (TDO) is a tetrameric enzyme that catalyses the oxidative cleavage of l-tryptophan (l-Trp) to N-formylkynurenine by the addition of O(2) across the 2,3-bond of the indole ring. This reaction is the first and rate-limiting step in the kynurenine pathway in mammals. In the present study, we measured the conformational changes in the haem pocket of recombinant human TDO (rhTDO) in ferric form that are induced by l-Trp binding using both resonance Raman and optical absorption spectroscopies. The deconvolution analysis of the haem Raman bands at various concentrations of l-Trp revealed that the wild-type enzyme exhibits homotropic cooperativity in l-Trp binding, which was confirmed by a change in the optical absorption spectra. Mutation analysis showed that the Y42F mutant abolished the cooperative binding, and that the H76A mutant considerably reduced the catalytic activity. These data and the inter-subunit contacts reported in the bacterial TDO structure suggest that the Y42 of rhTDO is responsible for the cooperative binding of l-Trp by participating in the active site of the adjacent subunit.
Fukumura
Eiko
E
Biometal Science Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo, Japan.
Sugimoto
Hiroshi
H
Misumi
Yuko
Y
Ogura
Takashi
T
Shiro
Yoshitsugu
Y
D020134:Catalytic Domain; D006801:Humans; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity; D014364:Tryptophan / Q000378:metabolism* / Q000494:pharmacology; D014366:Tryptophan Oxygenase / Q000737:chemistry / Q000378:metabolism*; D016959:Xanthomonas campestris / Q000201:enzymology
D050505:Mutant Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D014364:Tryptophan; D007501:Iron; D014366:Tryptophan Oxygenase