Structure-function relationships of rat hepatic tryptophan 2,3-dioxygenase: Identification of the putative heme-ligating histidine residues
Structure-function relationships of rat hepatic tryptophan 2,3-dioxygenase: Identification of the putative heme-ligating histidine residues
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DOI：
10.1006/abbi.2001.2420
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发表时间：
2001-08-01
影响因子：
3.9
通讯作者：
Correia, MA
中科院分区：
文献类型：
--
作者：
Dick, R;Murray, BP;Correia, MA
肝脏胞质酶色氨酸2,3-二加氧酶（TDO）催化L-色氨酸的氧化为甲酰基硝酸，并控制色氨酸的生理通量进入血清素能和kynureninic途径。该血蛋白酶由等效质量的四个非共价亚基组成，每个分子中包含两个血红素部分。电子顺磁共振分析表明，组二氮与血红素结扎有关[Henry等，（1976）J。Biol。化学251，1578]，但他的残留物的身份尚不清楚。为了表征酶的活性位点，我们用ALA代替了他在大鼠TDO亚基中的12个残基中的每个残基，以确定它们在血红素结合中的相对重要性。大鼠肝脏蛋白与其他生物体的已知或假定TDO序列的序列比对表明，他的四个残基在真核生物中是保守的，其中两个在原核生物中也是保守的。我们的发现表明，进化保存的76和328个残基的替代导致TDO活性的显着降低，而真核生物保守的HIS70的替换导致相对于野生型酶的HIS70的降低。另一方面，替代其他真核生物保守的HIS273残基，同时影响酶的相对表达，对其特定活性几乎没有影响。尺寸排斥分析表明，His76Ala和His328ala突变体几乎没有或没有血红素，这表明这些可能是将假体血红素部分连接的关键残留物。这些残留物是由同一TDO亚基提供的还是其他TDO亚基提供的尚待确定。 （c）2001学术出版社。
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. (C) 2001 Academic Press.