Structure of Pyridoxal Kinase from Sheep Brain and Role of the Tryptophanyl Residues
Structure of Pyridoxal Kinase from Sheep Brain and Role of the Tryptophanyl Residues
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DOI：
10.1023/a:1021079110358
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发表时间：
1999-04
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影响因子：
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通讯作者：
B. Maras;S. Valiante;S. Orrù;M. Simmaco;D. Barra;J. Churchich
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作者：
B. Maras;S. Valiante;S. Orrù;M. Simmaco;D. Barra;J. Churchich
羊脑吡哆醛激酶的一级结构已通过直接化学和物理方法测定。该酶含有 312 个氨基酸残基，N 末端有一个乙酰化蛋氨酸，分子量为 34,861 Da。多肽链第 52 和 244 位的两个色氨酸残基所发挥的功能作用已通过荧光光谱法进行了研究。色氨酸残基没有完全暴露于快速松弛溶剂中，并且碰撞猝灭剂很难接近它们。用 NBS 进行化学修饰会消除激酶的催化活性。羊脑酶的氨基酸序列与最近报道的人吡哆醛激酶显示出高度相似性（86.2%同一性）[Hanna, Turner, and Kirkness, (1997), J.生物。化学272, 10756–10760]。将哺乳动物蛋白与从 Swiss-Prot 数据库检索到的细菌和酵母推定吡哆醛激酶进行比较，显示总体相似度较低。特别是，假定的 ATP 结合结构域是保守的，而在吡哆醛底物的结合中似乎至关重要的区域则不然。因此，将细菌和酵母 cDNA 推导的蛋白质指定为吡哆醛激酶时应谨慎对待。
The primary structure of sheep brain pyridoxal kinase has been determined by direct chemical and physical methods. The enzyme contains 312 amino acid residues with an acetylated methionine at the N-terminus, yielding a molecular mass of 34,861 Da. The functional role played by the two tryptophanyl residues in positions 52 and 244 of the polypeptide chain has been investigated by fluorescence spectroscopy. The tryptophanyl residues are not completely exposed to the rapidly relaxing solvent and they are poorly accessible to collisional quenchers. Chemical modification with NBS abolishes the catalytic activity of the kinase. The amino acid sequence of the sheep brain enzyme shows high similarity (86.2% identity) with the human pyridoxal kinase recently reported [Hanna, Turner, and Kirkness, (1997),J. Biol. Chem.272, 10756–10760]. Comparison of the mammalian proteins with bacterial and yeast putative pyridoxal kinases retrieved from the Swiss-Prot data bank shows a low degree of overall similarity. In particular, the putative ATP-binding domain is conserved, whereas the region that appears to be crucial in the binding of the pyridoxal substrate is not. Thus, the assignment of the bacterial and yeast cDNA-deduced proteins as pyridoxal kinases should be taken with caution.