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Journal of Biochemistry Advance Access originally published online on January 17, 2009
Journal of Biochemistry 2009 145(4):505-515; doi:10.1093/jb/mvp002
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Cooperative Binding of L-Trp to Human Tryptophan 2,3-Dioxygenase: Resonance Raman Spectroscopic Analysis

Eiko Fukumura1,2, Hiroshi Sugimoto1,*, Yuko Misumi3, Takashi Ogura3,4 and Yoshitsugu Shiro1

1Biometal Science Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo; 2Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka; 3Department of Life Science; and 4Picobiology Institute, Graduate School of Life Science, University of Hyogo, Koto 3-2-1, Kamigori-cho, Ako-gun, Hyogo, Japan

*To whom correspondence should be addressed: Tel: +81-791-58-2817, Fax: +81-791-58-2818, E-mail: sugimoto{at}spring8.or.jp

Received November 8, 2008; Accepted January 6, 2009


  Abstract

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 O2 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.

Key Words: heam, allosteric regulation, tryptophan, Raman spectra

Abbreviations: CT1, charge transfer band; IDO, indoleamine 2,3-dioxygenase; L-Trp, L-tryptophan; Mb, myoglobin; rhTDO, recombinant human TDO; TDO, tryptophan 2,3-dioxygenase; xTDO, Xanthomonas campestris TDO


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