Structure--function relationships of rat hepatic tryptophan 2,3-dioxygenase: identification of the putative heme-ligating histidine residues.
The liver cytosolic enzyme tryptophan 2,3-dioxygenase (TDO) catalyzes the oxidation of L-tryptophan to formylkynurenine and controls the physiological flux of tryptophan into both the serotonergic and kynureninic pathways. This hemoprotein enzyme is composed of four noncovalently bound subunits of equivalent mass and contains two heme moieties per molecule. Electron paramagnetic resonance analyses have indicated that a histidyl nitrogen is involved in heme ligation [Henry et al., (1976) J. Biol. Chem. 251, 1578], but the identity of the His residue(s) is unknown. In an attempt to characterize the active site of the enzyme we have substituted each of the 12 His residues in the rat TDO subunit with Ala, to determine their relative importance in heme binding. Sequence alignment of the rat liver protein with that of known or putative TDO sequences from other organisms reveals that four of the His residues are conserved in eukaryotes, two of which are also conserved in prokaryotes. Our findings indicate that replacement of the evolutionarily conserved His 76 and 328 residues resulted in a dramatic reduction of TDO activity, whereas that of the eukaryotically conserved His70 resulted in a significant reduction relative to that of the wild-type enzyme. On the other hand, replacement of the other eukaryotically conserved His273 residue, while affecting the relative expression of the enzyme, had little effect on its specific activity. Size-exclusion analyses revealed that the His76Ala and His328Ala mutants retained little or no heme, suggesting that these may be key residues in ligating the prosthetic heme moieties. Whether these His residues are both provided by the same TDO subunit or a different TDO subunit remains to be determined.
Dick
R
R
Department of Cellular and Molecular Pharmacology, the Liver Center, University of California, San Francisco, California, 94143, USA.
Murray
B P
BP
Reid
M J
MJ
Correia
M A
MA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers / Q000235:genetics; D019143:Evolution, Molecular; D006418:Heme / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014366:Tryptophan Oxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D008024:Ligands; D006418:Heme; D006639:Histidine; D014366:Tryptophan Oxygenase